
==== Front
Transl Oncol
Transl Oncol
Translational Oncology
1936-5233
Neoplasia Press

S1936-5233(24)00230-4
10.1016/j.tranon.2024.102103
102103
Original Research
Combining Mitomycin C with inhibition of BAD phosphorylation enhances apoptotic cell death in advanced cervical cancer
Wang Liqiong a1
Zhang Xi bc1
Chen Shu cd1
Ye Qiuhua cd
Basappa Basappa e
Zhu Tao bfg
Lobie Peter E. pelobie@sz.tsinghua.edu.cn
bcd⁎
Pandey Vijay vijay.pandey@sz.tsinghua.edu.cn
cd⁎
a Department of Gynecology and Obstetrics, the University of Hong Kong-Shenzhen Hospital, Shenzhen, 518053, Guangdong, PR China
b Shenzhen Bay Laboratory, Shenzhen 518055, Guangdong, PR China
c Precision Medicine and Healthcare Research Center, Tsinghua-Berkeley Shenzhen Institute, Tsinghua University, Shenzhen 518055, Guangdong, PR China
d Institute of Biopharmaceutical and Health Engineering, Tsinghua Shenzhen International Graduate School, Tsinghua University, Shenzhen 518055, PR China
e Laboratory of Chemical Biology, Department of Studies in Organic Chemistry, University of Mysore, Manasagangotri, Mysore, 570006 Karnataka, India
f Department of Oncology, The First Affiliated Hospital of USTC, Center for Advanced Interdisciplinary Science and Biomedicine of IHM, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei 230027, Anhui, PR China
g Key Laboratory of Immune Response and Immunotherapy, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei 230027, Anhui, PR China
⁎ Corresponding author. pelobie@sz.tsinghua.edu.cnvijay.pandey@sz.tsinghua.edu.cn
1 These authors contributed equally to this work.

24 8 2024
11 2024
24 8 2024
49 1021036 4 2023
3 7 2024
11 8 2024
© 2024 Published by Elsevier Inc.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Highlights

• MMC treatment of cervical carcinoma (CC) cell lines stimulated phosphorylation of BAD at serine 99 (pBADS99) and the pBADS99/BAD ratio was positively correlated with MMC IC50.

• Mutation of BAD to BADS99A resulted in enhanced CASPASE3/7 activity and apoptosis in CC cell lines.

• A functional synergy using a treatment combination of MMC and a pBADS99 inhibitor (NPB) was observed in cell line and patient-derived models of CC.

Objective

Mitomycin C (MMC), a DNA-damaging chemotherapeutic, is commonly used clinically for recurrent cervical carcinoma (CC), either alone or in combination. MMC generates DNA damage resulting in CC cell death yet also induces increased AKT-BAD phosphorylation associated with drug resistance and reduced clinical benefit. The present study evaluates the efficacy of combined MMC and a BAD phosphorylation inhibitor in CC.

Methods

The association and function of phosphorylation of BAD on serine 99 (pBADS99) for cell survival of both MMC-resistant or sensitive-CC cells was explored. BAD was mutated to BADS99A to examine the requirement of BADS99 for CC cell survival and a novel small-molecule inhibitor of pBADS99 was utilized. Cell proliferation, survival, foci formation, and patient-derived organoids (PDOs) assays were utilized to determine efficacy, synergy and related mechanisms.

Results

MMC IC50 was positively correlated to the cell line pBADS99/BAD ratio. Increased BADS99 phosphorylation was observed in both MMC-sensitive or -resistant CC cells after MMC treatment. Inhibition of pBADS99 in CC cell lines produced synergistic apoptosis through BAD-mediated apoptotic pathways and enhanced DNA damage in response to MMC. The concurrent use of pharmacological inhibition of pBADS99 and MMC was synergistic, resulting in diminished cell viability and inducing apoptotic cell death in MMC-sensitive and -resistant CC cell lines or patient-derived organoids.

Conclusion

A combination of MMC with inhibition of BAD phosphorylation potentiated efficacy compared to single agent treatment. The potential further development of such strategies may provide outcome benefits to patients with CC.

Keywords

BAD phosphorylation
Mitomycin C
Apoptosis
cervical cancer
N-cyclopentyl-3-((4-(2,3-dichlorophenyl) piperazin-1-yl) (2-hydroxyphenyl) methyl) benzamide (NPB)
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pmcIntroduction

Despite the recent advancement in the screening, detection, and early diagnosis of cervical cancer (CC), >340,000 women died from CC worldwide in 2020 [1]. The median 5-year survival rate of CC patients approximates 66 percent (%). Late-stage diagnosis and dissemination of CC to surrounding tissues, regional lymph nodes, and/or distant organs decreases the 5-year patient's survival rate to between 17 and 58 % [1]. Patients with stage III to IVB exhibit a recurrence rate as high as 70 % [1]. Over recent decades, DNA damaging therapy utilizing chemotherapeutics such as cisplatin, paclitaxel, gemcitabine, topotecan, 5-fluorouracil, or mitomycin C (MMC), combined with radiotherapy, has produced improved survival outcomes for late-stage CC [2]. Nevertheless, the efficacy of DNA damaging-based therapies is limited by toxicities and the acquisition of therapy resistance [3], particularly associated with a suboptimal regression during radiotherapy of CC patients [4]. MMC, an FDA-approved DNA alkylating agent, has been widely used in CC as a constituent of combination therapy. As a potent DNA cross-linker, MMC generates DNA strand breaks in replicating cells, modulating several DNA-double strand break (DSB) repair proteins, and cell cycle regulatory effectors [5], and leads to pronounced induction of the mitochondrial-dependent intrinsic apoptotic pathway [6]. Combination therapy comprising MMC has improved animal model outcomes and shown additional survival benefits for CC patients [7]. However, the clinical effectiveness of MMC is often limited by the emergence of drug-resistant cancer cells [8]. Therefore, developing novel first-line combinatorial strategies to enhance the efficacy of DNA-damaging chemotherapy with limited toxicity and reduced therapy resistance is an unmet need for CC patient treatment.

Activation of the PI3K-AKT and MAPK signaling pathways has been positively correlated with increased resistance to radiation and genotoxic drugs and dissemination of CC [9]. Activated PI3K/AKT and MAPK pathways also increase cancer cell proliferation and resistance to MMC [10]; and an AKT phosphorylation inhibitor re-sensitizes the cancer cell response to MMC [10]. MMC has been observed to activate the RAS/MAPK and PI3K/AKT pathways [10], which limits the drug response [11]. BAD acts downstream of PI3K/AKT/mTOR and RAS/MAPK pathways through phosphorylation at S75 predominantly via p44/42 MAP kinase pathway and S99 predominantly via AKT/p70S6K; and dephosphorylated BAD promotes apoptosis by interacting with anti-apoptotic proteins, releasing apoptotic proteins like BAX and BAK [12]. Elevated pBAD/BAD ratios have been reported to be significantly associated with drug resistance, and prognosis in various malignancies, including CC [13]. For example, higher levels of phosphorylated BAD are reported to be associated with acquired resistance to radiotherapy in CC [14]. Hence, as a common downstream effector of the PI3K/AKT/mTOR and the RAS/MAPK pathways, pharmacological inhibition of BAD phosphorylation could be therapeutically beneficial as a combination strategy with MMC to promote apoptotic cell death and assist to obviate therapy-associated resistance in CC.

A small molecule (NPB) that inhibits BAD phosphorylation at S99, independent of kinase activity, and promotes apoptosis of cancer cells has been recently described [15]. This study evaluated the therapeutic synergy of a combination of NPB and MMC for efficacy in advanced CC using in vitro and ex vivo preclinical models.

Materials and methods

Cell culture and reagent

SiHa, C-33A, Ca-Ski, and HT3 cell lines were sourced from Procell Life Science & Technology Co. Ltd (Wuhan, China). The cell line clinical characteristics and molecular profiling are tabulated in Table S1 of the supplementary material. MMC was purchased from Selleckchem (Houston, TX, USA). The BADS99 inhibitor, N-cyclopentyl-3-((4-(2,3-dichlorophenyl) piperazin-1-yl) (2-hydroxyphenyl) methyl) benzamide (NPB), was synthesized following previously published protocols [15]. All erxperiments were performed in medium containing 2 % FBS unless otherwise indicated.

Construction of hBADS99A plasmid and transient transfection

A nucleotide mutation from TCG into GCC was introduced to the coding sequence (CDS) to generate BADS99A in which S99 was replaced with Ala. hBADS99A cDNA was then sub-cloned into pcDNA3.1(+) plasmid vector (with Nhe I (R0131, New England Biolabs (Beijing) Ltd., PRC) and EcoR I(R0101, New England Biolabs (Beijing) Ltd., PRC)restriction endonuclease. The CDS of hBADS99A was sequenced by Sangon Biotech (Shanghai, PRC), and the sequence is listed in Table S2. The construct was designated as pcDNA3.1-hBAD-S99A. Transient transfection of cell lines was achieved with FuGENE® 6 Transfection Reagent (E2692, Promega, Madison, WI, USA) following the manufacturer's guidelines and a previously described method [15].

Western blot (WB) analysis

WB analysis was carried out according to previously published methods [15] utilizing primary antibodies and secondary antibodies (anti-rabbit, anti-mouse, and anti-goat horseradish peroxidase (HRP)-conjugated) listed in Table S3. Cells were lysed using RIPA buffer and resolved by SDS-polyacrylamide-gel electrophoresis. Visualization of blots was achieved using Clarity™ and Clarity Max™ Western ECL Blotting Substrates (BIO-RAD, USA).

Comet assay

Briefly, cells were treated with NPB at indicated concentrations for 48 h and then 1 × 103 cells were mixed with 0.7 % low-gelling agarose and layered as microgels on microscopic slides. The cells were then lysed in a solution containing 146 mM NaCl, 30 mM EDTA, pH7, 10 mM Tris–HCl, pH7 and 0.1 % N-lauroylsarcosine at 10 °C for 20 min. Electrophoresis was performed for 20 min at 0.46 V/cm. The results were visualized under a fluorescent microscope after staining of gels with SYBR green and quantified using the CometScore software, specialized for comet assay analysis.

BAD co-immunoprecipitation (co-IP)

The co-IP assay was performed using BAD-antibody conjugated magnetic beads from Universal IP/Co-IP Toolkit (Abbikine) following the manufacturer's guidelines (Cat #: KTD104-EN, Abbikine, China). Briefly, Protein A G-1 Magnetic Beads were conjugated with BAD-antibody (#9268, Cell Signaling Technology, Inc.) or Rabbit IgG after three washes with Wash Buffer. Cells were lysed by Non-Denaturing Lysis Buffer and centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant was collected, and Bicinchoninic Acid Assay detected the protein concentration. Protein samples were incubated with antibody-conjugated magnetic beads for 4 °C overnight. After washing, 1×SDS-PAGE Loading Buffer was added to protein-binding magnetic beads and mixed well, incubated at 100 °C for 5 min, then centrifuged at 800 rpm for 1 min. The supernatant was collected for subsequent SDS-PAGE and WB analysis.

Oncogenic and immunofluorescence (IF) analyses

The methods for AlamarBlue® cell viability, foci formation and ex vivo (3D Matrigel) culture were the same as those previously described [15], with all cell-based assays performed in media supplemented with 2 % FBS. The ApoTox-Glo™ Triplex Assay Kit (G6320, Promega, China) was used to evaluate cell viability, apoptosis, and cytotoxicity, following the same procedures as previously described [15] using a Tecan microplate reader. Annexin-V-FLUOS and PI-staining (Neobioscience, Shenzhen, China) were utilized to assess early- or late-apoptotic cells as previously described [15]. The LIVE/DEAD™ Cell Imaging Kit (ThermoFisher Scientific, USA) was utilized to visualize live or dead cells, following the methods described in previous publications [16]. Chou-Talalay combination index (CI) analysis was used to evaluate the synergy between agents, as previously reported [16,17] as described in the flow chart (Fig. S3). Immunofluorescence (IF) analyses were conducted using an inverted fluorescence microscope (Olympus IX73P1F, Japan) or confocal microscopy (C2+, Nikon, Japan) and followed previously described procedures [17]. The details of primary and secondary antibodies details are provided in Table S3. A mounting medium with DAPI (ab104193, Abcam) labeled the cell nuclei.

Patient-derived cancer cell (PDC) and organoid (PDO) culture and treatment

For the current study, approximately 1 g of patient-derived CC specimens were used to obtain PDCs and PDOs. The patient-derived CC specimens were acquired from the University of Hong Kong-Shenzhen Hospital (HKU-SZH) with written consent from patients and with the approval of the Ethical Committee of HKU-SZH, as stated in the Institutional Review Board Statement section. Patient-derived CC organoids were established and characterized as described in Supplementary Information (Supplementary methods) [16]. A pathologist diagnosed all pathologies, and the clinical information is provided in Fig. S6A. Immunofluorescence staining for VIMENTIN (VIM) and CYTOKERATIN 8 (CK8) was performed on PDOs to determine the epithelial content (Fig. S6C). Treatment was initiated on the third day following organoid formation, and the treatment medium was replaced every two days. The organoid expansion was evaluated by organoid counting under an inverted microscope [18]. PDOs were evaluated using the ApoTox-Glo Triplex Assay Kit for survival fraction (SF) and caspase3/7 activity.

Immunohistochemistry (IHC)

Organoids were fixed overnight in 4 % paraformaldehyde (PFA): PBS, followed by gradient dehydration in sucrose solutions of 10 %, 20 %, and 30 % (Sigma, St Louis, MO). The organoids were embedded in OCT cryo-embedding media (Tissue-Tek, US) and frozen at −80 °C. Slices of 5 µm were obtained using a freezing microtome (NX50, Thermo Fisher Scientific) and subjected to standard H&E staining. Images were captured using an inverted microscope (CX31, Olympus, Japan) [19]. Densitometric analysis of IHC was performed using ImageJ software (https://imagej.nih.gov/ij/).

Statistical analysis (SA) and reproducibility

GraphPad Prism 7.0 software was utilized for statistical analysis as described previously [15]. For subgroup analysis of in vitro assays, an unpaired two-tailed Student t-test was employed to determine statistical differences. p-values (<0.05, *; p < 0.01, **; and p < 0.001, ***) indicated significance levels. Unless stated otherwise, the numerical data are presented as mean ± SD. Different assays were performed independently at least three times and showed similar results.

Results

Higher pBADS99/BAD levels promote cell survival in CC cells

Using WB analysis, the levels of pBADS99, pAKTS473, and the expression of BAD, and AKT protein were determined in CC cell lines, namely, SiHa, C-33A, Ca-Ski, and HT3 (Table S1). These cell lines exhibited expression of BAD and AKT and phosphorylation of BADS99, and AKTS473, as demonstrated in Fig. 1A. After normalizing to BAD protein expression, MMC-most resistant SiHa cells (also radiation-resistant) [20] displayed a significantly higher pBADS99/BAD ratio compared to MMC-sensitive cell lines C-33A. HT-3 and Ca-Ski exhibited a medium ratio. For the pBADS75/BAD ratio, MMC-resistant SiHa exhibited a high ratio, HT-3 possessed a medium ratio, and C-33A and Ca-Ski showed low ratios. As the activated AKT pathway leads to BAD phosphorylation at Ser99, SiHa cells also exhibited significantly higher pAKTS473 levels compared to the other three cell lines (heatmap of Fig. 1A). To assess the significance of the pBADS99/BAD ratio in response to MMC in the CC cell lines, the pBAD/BAD ratio was plotted against MMC IC50 values using Pearson correlation analysis (Fig. 1A). A higher pBADS99/BAD ratio in CC cell lines was positively correlated with MMC IC50 (p < 0.05).Fig. 1 Higher pBADS99/BAD promotes cell survival in MMC-resistant CC cells. (A) Left: Western blot (WB) analysis was performed to evaluate pBADS99, pBADS75 and pAKTS473 levels and AKT and BAD protein expression in SiHa, C-33A, Ca-Ski, and HT3 cell lines. Right: Heatmap represents the densitometry analysis of BAD and AKT expression; and the ratio of pBADS99/BAD, pBADS75/BAD, and pAKTS473/AKT in four CC cell lines. The below graph shows the Pearson correlation of pBADS99/BAD with Mitomycin C (MMC) IC50 values in the CC cell lines. (B) WB evaluation of the protein activities and levels of different proteins in SiHa cells following treatment with Mitomycin C (MMC) of increasing concentrations. (C) SiHa, C-33A, Ca-Ski, and HT3 cell lines were transiently transfected with an S99-mutated human (h) BAD (hBADS99A, with Serine 99 residue mutated to alanine) cDNA and wild type (WT) hBAD (hBADWT) cDNA as control. Forced expression of hBADS99A was confirmed using WB analysis. (C) The ApoTox-Glo Triplex Assay Kit was used to determine CASPASE 3/7 activity and (D) cell viability of SiHa, C-33A, Ca-Ski, and HT3 cell lines was measured 48 h after transfection (n = 3). Columns are the mean of triplicate experiments; bars, ±SD. *p < 0.05, ** p < 0.01, *** p < 0.001. Columns are the mean of triplicate experiments; bars, ±SD. *p < 0.05, ** p < 0.01, *** p < 0.001.

Fig. 1

Furthermore, exposure of SiHa or C-33A cells to MMC resulted in a dose-dependent increase in pAKTT308/AKT, pAKTS473/AKT, and pBADS99/BAD ratios compared to vehicle-treated cells (Figs. 1B and S1A), indicating that an elevated pBADS99/BAD ratio may contribute to MMC resistance in CC cells.

To determine the functional contribution of the pBADS99/BAD ratio in regulating CC cell survival, SiHa, C-33A, Ca-Ski, and HT3 cells were transiently transfected with an S99-mutated human (h) BAD (hBADS99A, with Serine 99 residue mutated to alanine, preventing phosphorylation at Ser99) cDNA and hBAD (wild type) cDNA as control. Forced expression of hBADS99A in SiHa cells significantly decreased the pBADS99/BAD ratio (Fig. 1C). Additionally, hBADS99A expression in SiHa cells significantly (p < 0.001) increased CASPASE3/7 activity (Fig. 1D) and reduced cell viability (Fig. 1E) compared to hBAD-transfected SiHa cells. Similar changes in pBADS99/BAD levels, CASPASE3/7 activity, and cell viability were observed in C-33A, Ca-Ski, or HT3 cells after hBADS99A expression, indicative that reduced levels of phosphorylated BADS99 promote apoptosis in CC cells.

NPB increases apoptosis in MMC-resistant CC cells

Using western blot analysis, the efficacy of a small molecule pBADS99 inhibitor, NPB, was determined in SiHa and C-33A cells (Figs. 2A and S2A). NPB-treated SiHa cells exhibited a dose-dependent decreased ratio of pBADS99/BAD. In contrast, no significant change was observed in the pBADS75/BAD or BAD/β-ACTIN levels in SiHa cells after treatment with NPB. The levels of BCL-2, BCL-W, or BCL-XL protein were not altered in SiHa cells after treatment with NPB. SiHa cells treated with NPB also produced a dose-dependent increase in the levels of cleaved- poly (ADP-ribose) polymerase (PARP) and γH2AX indicating that NPB treatment promoted DNA damage in SiHa cells as expected [21]. Also, NPB treatment increased cleaved CASP3, cleaved-CASP7, and TP53 levels in SiHa cells, indicating a caspase-dependent intrinsic apoptosis [22]. Similar directional changes were observed in C-33A cells. To further investigate the effect of NPB on DNA damage, a comet assay was performed on SiHa and C-33A cell lines. Increasing doses of NPB significantly increased the area and length of the DNA tail in both cell lines, indicating increased DNA damage with NPB treatment (Figs. 2B and C).Fig. 2 The pBADS99 inhibitor, NPB, stimulates BAD-mediated apoptosis in MMC-resistant CC cells. (A) Western blot (WB) analysis was performed to evaluate pBADS99, pBAD75, BAD, BCL-2, BCL-W, BCL-XL, Cleaved PARP, γH2AX, CASPASE3(CASP3), Cleaved CASP3, CASPASE7(CASP7), Cleaved CASP7 and TP53 in SiHa cell lines treated with NPB at the indicated concentrations for 48 h. (B) A comet assay was performed on SiHa cells treated with NPB at the specified concentrations for 48 h. DNA damage was estimated by measuring the DNA tail area as a percentage (%) of the total DNA area and the comet tail length (from the center of the DNA head to the end of the DNA tail). The larger the DNA tail area% or the longer the DNA tail length indicates more significant damage. The captured images were magnified at ×200, with a scale bar of 100 μm. (C) Effect of 10 µM NPB, 200 nM Mitomycin C (MMC) and combination (Com) treatment for 48 h on co-immunoprecipitation (co-IP) of BAD and BCL-2, BCL-XL or BCL-W in SiHa cells as described in the methodology. The heavy chain is denoted as HC. Columns are the mean of triplicate experiments; bars, ±SD. *p < 0.05, ** p < 0.01, *** p < 0.001.

Fig. 2

Next, using co-IP analysis, the interaction of BAD with BCL-2, BCL-XL or BCL-W was determined in SiHa and C-33A cells after NPB, MMC, or Combination (Com) treatment. Treatment of SiHa cells with NPB, MMC, or Com did not significantly alter expression levels of BAD, BCL-XL, or BCL-W, as demonstrated by WB analysis of input-lysate, whereas BCL-2 expression was decreased with MMC; neither NPB nor Combination influence expression of BCL-2. BAD immunoprecipitated from NPB-, MMC- or Com-treated SiHa cells exhibited no significant difference in levels compared to immunoprecipitated BAD (BAD-IP) from control cells when normalized to IgG-HC. However, the BAD-IP from NPB- or Com-treated SiHa cells exhibited significantly (p < 0.001) increased levels of BCL-2, BCL-XL or BCL-W protein compared to the BAD-IP from control cells. In contrast, MMC increased levels of BCL-XL and BCL-W but not BCL-2 compared to the BAD-IP from control cells (Figs. 2B and S1C). Therefore, treatment of SiHa cells with NPB increased the interaction of BAD with BCL-2, BCL-XL and BCL-W (Fig. 2C). Similar directional changes were observed in co-IP assays in C-33A cells (Fig. S2D).

NPB and MMC synergistically reduce CC cell survival

SiHa cells exhibited the most resistance to MMC, an approximate 70-fold higher IC50 value of MMC compared to most MMC-sensitive C-33A as observed using cell viability assays (Fig. 3). IC50 values of MMC in HT-3 and Ca-Ski are between them. Using Pearson correlation analysis (Fig. 1A), the pBAD/BAD ratio in these four CC cells was positively correlated with MMC IC50 (p < 0.05) as shown in Fig. 1A. Exposure of the four CC cell lines to NPB significantly decreased cell viability compared to DMSO-treated CC cells. Using Chou-Talalay analysis, it was next examined whether NPB synergizes with MMC in SiHa, C-33A, Ca-Ski, and HT3 cell lines. NPB exhibited synergy with MMC in all four CC cell lines, based on combination index (CI) analysis (Fig. 3). The median combination index value for NPB-MMC doses was below one, indicating a synergistic effect (Fa 0.5–0.95) across the four CC cell lines. Using a cell viability assay, it was observed that combined treatment with NPB (1 µM) and MMC significantly enhanced the efficacy of MMC compared to MMC treatment alone, as demonstrated by dose-response curve (DRC) analyses (Fig. 3). Notably, combined NPB (1 µM)-MMC treatment of SiHa cells resulted in a ∼20-fold decrease in MMC IC50 (400±90 nM) compared to MMC alone (20±10 nM). In C-33A cells, the combined NPB (1 µM)-MMC treatment decreased MMC IC50 by ∼55-fold (5.86±1.4 nM vs. 0.1 ± 0.03 nM), in HT3 cells by ∼9-fold (144.85±6.72 nM vs. 16.48±2.49 nM), and in Ca-Ski cells by ∼6-fold (202.05±3.18 nM vs. 35.91±11.33 nM) compared to MMC treatment alone (Fig. 3). Thus, the combined NPB-MMC treatment synergistically reduced CC cell survival.Fig. 3 NPB synergizes with MMC in CC cells to decrease cell survival.

The cell survival fraction (%) was measured using AlamarBlue® cell viability assay after 72 h of treatments. The synergistic effect of combined NPB-MMC at the indicated concentrations (Conc.) in SiHa, C-33A, Ca-Ski and HT3 cell lines was analyzed using Chou-Talalay analysis. The logarithmic combination index (CI) value corresponding to cell fraction affected (Fa) was determined using the CompuSyn software (http://www.combosyn.com) as described in the methodology. CI <1 (log10CI<0) indicates synergism. Dose-response curves (DRC) of MMC at indicated concentrations with or without 1 µM NPB showed a significant reduction in MMC IC50 in the presence of NPB. The arrow indicates fold reduction in MMC IC50 in the presence of NPB. The results were statistically significant (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 3

A combination of NPB and MMC synergistically decreases foci formation and growth in 3D Matrigel by inducing apoptotic death of CC cells

Foci formation assay showed that single-agent NPB or MMC or combined NPB-MMC treatment attenuated the capacity for foci formation on monolayer compared to vehicle-treated MMC-resistant SiHa and MMC-sensitive C-33A cells (Fig. 4A). Moreover, combined NPB-MMC treatment significantly abrogated the capacity for foci formation on monolayer compared to NPB or MMC alone (p < 0.001). Annexin V-PI FACS analysis demonstrated that either single drug or combined NPB-MMC treatment significantly increased the percentage of apoptotic cells compared to vehicle-treated cells. Moreover, combined NPB-MMC treatment produced a significantly increased population of apoptotic cells compared to cells treated with either NPB or MMC alone (Fig. 4B). Similar directional changes were also observed in foci formation, Annexin V-PI positive in Ca-Ski and HT-3 cell lines (Fig. S4A and B). It was observed that the treatment of SiHa cells with NPB resulted in a minor increase in the cell population in the G1-phase, accompanied by a decrease in the S-phase (p < 0.05) as compared to MMC-treated cells (Fig. S4C). No significant alterations in the sub-G1 or S-phase cell populations were observed in SiHa cells treated with either NPB-MMC combined or individually. Comparable changes in foci formation, Annexin V-PI positivity, and the sub-G1/S-phase cell population of MMC-sensitive C-33A cells were observed after administering either a single drug or combined NPB-MMC.Fig. 4 NPB synergizes with MMC in CC cells to decrease foci formation in monolayer culture and growth in 3D Matrigel by promoting apoptotic death. (A) The effect of NPB (5 µM), MMC (100 nM MMC in MMC-resistant SiHa cells; 1 nM MMC in MMC-sensitive C33A cells), or their combination for fourteen days on foci formation was evaluated in SiHa and C-33A cell lines using crystal violet staining. Absorbance was measured at 570 nm using a microplate reader. The corresponding quantification is shown on the right side. The statistical change between treatment groups was analyzed using an unpaired two-tailed Student t-test. (B) Flow cytometry analysis was performed to measure apoptotic cell death using Annexin-V and propidium iodide staining in SiHa and C-33A cell lines after treatment with NPB (10 µM), Mitomycin C (MMC) (200 nM MMC in MMC-resistant SiHa cells; 10 nM MMC in MMC-sensitive C33A cells), or a combination of NPB and MMC (Com) after 72 h incubation as described in materials and methods (n = 3). The results show that the combination of NPB and MMC significantly decreased the survival fraction, increased Annexin-V positive cells, and decreased live cell colonies in SiHa and C-33A cell lines compared to treatment with either drug alone. The corresponding quantification is shown on the right side. The statistical change between treatment groups was analyzed using an unpaired two-tailed Student t-test. (C) Live colonies and dead cell debris were visualized using Calcein-AM and BO-BO-3 Iodide staining, respectively, in ex vivo cultured SiHa and C-33A cell lines in 3D Matrigel after exposure to NPB (5 µM), MMC (100 nM MMC for MMC-resistant SiHa cells; 1 nM MMC for MMC-sensitive C33A cells), or their combination. The ApoTox-Glo Triplex Assay Kit was used to evaluate CASPASE 3/7 activity and cell viability in SiHa and C-33A cells treated with NPB, MMC, or their combination for fourteen days in Matrigel culture. The ApoTox-Glo assay demonstrated reduced cell viability and elevated CASPASE 3/7 activity in cells treated with the combination compared to single drug treatment. The mean of triplicate experiments and standard deviation bars were plotted for each condition, and statistical significance was determined using an unpaired two-tailed Student t-test. Columns are the mean of triplicate experiments; bars, ±SD. *p < 0.05, ** p < 0.01, *** p < 0.001.

Fig. 4

In addition, pre-existing colonies of SiHa or C-33A CC cells grown in 3D Matrigel were subjected to a single agent or combined NPB-MMC treatment. In the case of SiHa cells, treatment with either NPB or MMC alone or combined NPB-MMC resulted in a noticeable increase in red fluorescence (indicating loss of plasma membrane integrity as stained by ethidium homodimer-1) and a decrease in green fluorescence (indicating intercellular esterase activity as stained by Calcein-AM) as compared to vehicle-treated SiHa colonies in 3D Matrigel culture (Fig. 4C, left panel). ApoTox-Glo assays were performed to measure the protease activity in live and CASPASE 3/7 activity of pre-grown SiHa cell colonies cultured in Matrigel to assess the affected cell fraction. Combined NPB-MMC treatment of SiHa cell colonies showed significantly (p < 0.001) increased CASPASE 3/7 activity and reduced cell viability as compared to MMC-resistant SiHa cell colonies treated with either NPB or MMC alone in Matrigel culture (Fig. 4C). The CASPASE 3/7 activity and cell viability exhibited similar directional changes in pre-existing C-33A colonies in Matrigel culture after treatment with either NPB or MMC alone or in combination (Fig. 4C, right panel).

NPB and MMC act synergistically in CC cells to induce DNA damage and increase BAD-mediated intrinsic apoptotic death

WB analysis was used to investigate the mechanism of the synergistic effect of NPB-MMC treatment of CC cell lines (see Figs. 5A and S5A). When SiHa cells were treated with NPB alone, the levels of pBADS99 normalized to BAD protein were significantly decreased, whereas no significant changes were observed in BAD, pAKT (T308 & S473), or AKT levels when compared to vehicle-treated cells. In contrast, MMC treatment of SiHa cells increased pAKTT308, pAKTS473, and pBADS99 normalized to AKT and BAD protein levels, respectively. When SiHa cells were treated with a combination of NPB and MMC, the levels of pBADS99/BAD were significantly reduced when compared to cells treated with NPB or MMC alone. No significant changes in pAKTT308/pAKTS473 or AKT protein levels were observed in MMC-resistant SiHa cells treated with combined NPB-MMC. pAKT (T308 & S473) and pBADS99 levels showed similar directional changes in C-33A, Ca-Ski and HT3 cell lines after treatment with NPB alone or combined with MMC (Fig. S5A).Fig. 5 NPB synergizes with Mitomycin C (MMC) in CC cells to stimulate DNA damage and enhance BAD-mediated apoptotic cell death. (A) Western blot (WB) analysis was performed to evaluate pBADS99, BAD, pAKTT308, pAKTS473, AKT, γH2AX and cleaved PARP in SiHa and C-33A cell treated with NPB (10 µM), MMC (200 nM MMC in MMC-resistant SiHa cells; 10 nM MMC in MMC-sensitive C33A cells), or a combination of both for 48 h. (B) Representative immunofluorescence images of γH2AX were captured using a confocal microscope in SiHa and C-33A cell lines treated with (10 µM), Mitomycin C (MMC) (200 nM MMC in MMC-resistant SiHa cells; 10 nM MMC in MMC-sensitive C33A cells), or a combination of both for 24 h. The scale bars in the images were denoted as 50 µm.

Fig. 5

γH2AX has been identified as a critical DNA damage marker in CC cells [23]. WB and IF analyses showed that treatment of SiHa cells with either NPB or MMC led to increased levels of γH2AX protein compared to control cells (Figs. 5A and B and S5C). Notably, MMC treatment resulted in higher levels of γH2AX protein than NPB treatment alone. The combined treatment of NPB and MMC in SiHa cells significantly increased γH2AX levels compared to either NPB or MMC treatment alone (Fig. 5A). Similarly, the combined treatment of MMC-resistant SiHa cells with NPB and MMC resulted in a markedly increased number of γH2AX foci in nuclei (p < 0.01) compared to NPB or MMC treatment alone (Fig. S5C). C-33A cells exhibited similar directional changes in the levels of γH2AX protein and foci in the nuclei after treatment with NPB, MMC, or a combination of both (Figs. 5A and B and S5C). Ca-Ski and HT3 cell lines showed similar directional changes in the levels of γH2AX protein (Fig. S5A and B).

Additionally, SiHa cells treated with NPB or MMC alone resulted in increased levels of cleaved-PARP protein compared to control cells. Combined NPB-MMC treatment of SiHa cells showed a marginally increased cleaved-PARP protein level compared to vehicle or NPB or MMC treatment alone (Fig. 5A). Similar changes were observed in the levels of cleaved-PARP protein in C-33A, Ca-Ski and HT3 cell lines treated with NPB, MMC, or a combination of both (Fig. S5A and B). Therefore, inhibition of pBADS99 synergizes with MMC in CC cells to enhance DNA damage and intrinsic apoptotic cell death.

Combined NPB-MMC treatment stimulates apoptotic cell death in CC PDOs

PDOs are a validated preclinical model to evaluate anti-cancer drug efficacy [24,25]. Herein, three CC PDOs, PDO1–3, were derived from resected primary CC and established as described in methodology section 2.7 (above). The CC cases were diagnosed at locally advanced stages (FIGO IIA1, FIGO IIIC and FIGO IIA1) (Fig. S6A), which are not suitable for hysterectomy, radical trachelectomy, or exenteration alone [26].

The functional efficacy of combined NPB-MMC treatment of CC PDOs was determined in 3D Matrigel (ex vivo organoid culture). Treatment of CC PDO1 with NPB or MMC resulted in increased CASPASE 3/7 activities and decreased cell survival compared to control CC PDO1. When combined, NPB-MMC treatment of CC PDO1 produced significantly (p < 0.001) higher CASPASE 3/7 activities and reduced cell survival compared to CC PDO1 treated with NPB or MMC alone (Figs. 6A and S3B). Additionally, combined NPB-MMC treatment of CC PDO1 resulted in significantly reduced organoid growth compared to NPB or MMC-treated CC PDO1 (Figs. 6A and S6B). Similarly, CC-PDO2 and -PDO3 exhibited comparable changes in CASPASE 3/7 activities, cell survival, and organoid growth after treatment with NPB or MMC alone or combined (Figs. 6A and S6B).Fig. 6 Combined NPB-MMC treatment stimulates apoptotic cell death in patient-derived organoids (PDO) of CC. (A) The effect of 10 µM NPB, 10 nM Mitomycin C (MMC), or combination (Com) treatment for seven days on three CC PDOs was examined using the ApoTox-Glo assay. The growth of organoids was assessed by calculating the average diameter of the organoid on the seventh day, and images illustrating primary tumor cells in monolayer culture and organoid growth on the seventh day were presented. The statistical change between treatment groups was analyzed using an unpaired two-tailed Student t-test. All images were captured at ×200 magnification, and the scale bars represent 100 μm. (B) Haematoxylin & Eosin (H&E) was used to show organoid structure and Immunohistochemistry (IHC) was used to evaluate pBADS99, BAD, KI67, and TdT-mediated dUTP Nick-End Labeling (TUNEL) positive staining in PDOs treated with 10 µM NPB, 10 nM MMC, or Com for nine days, as described in materials and methods. The bar chart presented in the below panel displays the number of cells with positive staining for the respective treatment groups (n = 3). All images were captured at ×200 magnification, and the scale bars represent 20 μm. The statistical change between treatment groups was analyzed using an unpaired two-tailed Student t-test.

Columns are the mean of triplicate experiments; bars, ±SD. *p < 0.05, ** p < 0.01, *** p < 0.001.

Fig. 6

To further confirm the effect of NPB-MMC treatments, a histological analysis of CC PDO2 and PDO3 was performed (Fig. 6B). CC PDO2 organoids treated with NPB showed a significant reduction (p < 0.01) in pBADS99-positive cells compared to control CC PDO2. Conversely, treatment with MMC significantly (p < 0.01) increased pBADS99-positive cells compared to control CC PDO2. Treatment with a combination of NPB and MMC significantly reduced pBADS99-positive cells compared to treatment with NPB or MMC alone. No significant changes were observed in BAD-positive cells in PDO2 groups treated with either NPB or MMC or a combination of NPB and MMC. In addition, treatment of CC PDO2 with NPB or MMC resulted in decreased KI67-positive cells and increased apoptotic cells compared to control CC PDO2. Treatment with a combination of NPB and MMC resulted in significantly (p < 0.001) decreased KI67-positive cells and increased apoptotic cells compared to treatment with NPB or MMC alone (Fig. 6B). Similar directional changes were observed in pBADS99, BAD, and KI67; and apoptotic cell death of CC PDO3 after treatment with NPB or MMC alone or combined (Fig. S6D). These results suggest that NPB and MMC synergistically induce apoptotic cell death in CC PDOs cultured ex vivo.

Discussion

Despite MMC being a potent stimulator of apoptosis in CC cells, the emergence of resistance to MMC represents a significant challenge that leads to chemotherapeutic failure and a worse patient prognosis. The activation of PI3K/AKT/mTOR signaling is an alternative survival mechanism for the evasion of MMC-induced cell death, including in CC [9,10]. Indeed, activating compensatory survival pathways such as PI3K/AKT or MAPK have been positively correlated with reduced therapeutic efficacy. For that reason, a combined MMC-mTOR inhibitor approach produces synergistic effects on apoptosis through the decreased function of the S6K1–BAD–BAK pathway [27]. Phosphorylation of BAD serves as a core downstream effector of both PI3K and MAPK pathways [12]. Herein, it was also observed that MMC treatment produced a dose-dependent increase in BADS99 phosphorylation with an increased pBADS99/BAD ratio. Inhibition of pBADS99 with a small molecule (NPB) augmented MMC-induced apoptosis in SiHa cells resistant to MMC treatment. Also, combined NPB-MMC treatment increased levels of γH2AX, cleaved-CASP3/7, and cleaved-PARP protein in CC cell lines. A similar synergy was also observed in CC patient-derived organoids, wherein combined NPB-MMC treatment increased the activities of CASP3/7, enhanced apoptotic cell death, and reduced organoid growth. Thus, a combination treatment approach utilizing a pBADS99 inhibitor and MMC may provide a robust treatment strategy for CC explicitly resistant to MMC.

Notably, human papillomavirus (HPVs) (HPV16 and HPV18) has been etiologically associated with CC [28]. It has been reported that three oncoproteins, E5, E6, and E7, encoded by HPV, activated EGFR, MAPK, or PI3K/AKT/mTOR signaling cascades, resulting in the phosphorylation of BAD [29]. HPV-positive CC is recognized as more sensitive to DNA-damaging therapy compared to HPV-negative counterparts [30]. However, resistance to DNA-damaging therapy still exists, as feedback activation of the RAS/MAPK and/or PI3K/AKT/mTOR pathways has often been associated with therapy-resistant cancer [31]. Hence, as a core effector of both pathways, inhibition of the actions of phosphorylated BAD in cancer cells (herein with Ser99 mutated BAD or NPB) decreases cancer cell survival by promoting intrinsic apoptosis [15]; and more so in the presence of cytotoxic agents. Another example of synergy upon inhibition of BADS99 phosphorylation was observed in epithelial ovarian carcinoma (EOC) cells. The simultaneous use of NPB and cisplatin significantly inhibited the growth of EOC xenografts to a greater extent than NPB or cisplatin alone [32], partly by diminishing the population of stem cell-like cells in cisplatin-resistant EOC. Inhibition of BADS99 phosphorylation also synergizes with Poly (ADP-ribose) polymerase (PARP) inhibitors in ovarian [16] and endometrial carcinoma (EC) cells [17] and significantly improves the efficacy of PARP inhibition to reduce both cell line and patient-derived xenograft growth. Notably, combined inhibition of BADS99 and PARP in patient-derived xenografts exhibited a complete pathological response in some animals with prolonged host-animal survival outcomes. Inhibition of BADS99 phosphorylation enhances PARP inhibitor-induced DNA damage and impairs consequent DNA repair post-PARP inhibition, resulting in enhanced apoptotic cell death. This study also observed that treating CC cells with NPB resulted in elevated levels of γH2AX, which suggests a disruption in the DNA damage response of these cells. It is noteworthy that radiation therapy, which produces DNA damage, has been shown to induce phosphorylation of BAD, which contributes to therapy resistance [33]. The pBAD/BAD ratio determines cellular apoptosis or survival [12]. When the pBAD/BAD ratio decreases, it triggers permeabilization of the mitochondrial outer membrane through the BAK/BAX pathway, which causes the release of cytochrome-c into the cytoplasm, leading to the activation of caspase-dependent intrinsic apoptosis [34,35]. It was observed herein that combination treatment with NPB and MMC is associated with increased levels of cleaved-CASP3/7, resulting in increased CASPASE 3/7 activities and consequent apoptotic cell death. Hence, determining a pBAD/BAD ratio in CC may also serve as a prognostic marker for the efficacy of DNA-damaging-based treatment strategies.

Conclusions

The findings herein indicated that MMC treatment of CC cells promoted phosphorylation of BADS99, which enhanced cell survival with resultant chemoresistance. Combination treatment of CC cell lines with MMC and a BAD phosphorylation inhibitor produced DNA damage yet hindered feedback survival signals from the RAS/MAPK or PI3K/AKT/mTOR by inhibiting BADS99 phosphorylation. Consequently, enhancing caspase-dependent apoptotic cell death. The present study provides a promising strategy to improve the response of DNA-damaging agents in CC. Thus, inhibition of BAD phosphorylation in combination with DNA-damaging agent-based strategies in CC warrants further exploration.

CRediT authorship contribution statement

Liqiong Wang: Resources, Writing – review & editing. Xi Zhang: Investigation, Methodology, Resources, Writing – original draft, Writing – review & editing. Shu Chen: Methodology. Qiuhua Ye: Formal analysis, Investigation, Methodology, Software. Basappa Basappa: Methodology, Resources. Tao Zhu: Conceptualization, Writing – review & editing. Peter E. Lobie: Conceptualization, Funding acquisition, Project administration, Writing – review & editing. Vijay Pandey: Conceptualization, Data curation, Project administration, Writing – review & editing.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

V.P., B., and P.E.L. are listed as inventors on a patent application (and all derivatives) for NPB, which is used in this work (WO/2019/194520). P.E.L. is an equity holder in Sinotar Pharmaceuticals Ltd, which currently holds the license for this patent.

Appendix Supplementary materials

Image, application 1

Acknowledgments

This research was funded by the Shenzhen Key Laboratory of Innovative Oncotherapeutics (ZDSYS20200820165400003) (10.13039/501100010877 Shenzhen Science and Technology Innovation Commission ), China; Shenzhen Development and Reform Commission Subject Construction Project ([2017]1434 ), China; Overseas Research Cooperation Project (HW2020008) (Tsinghua Shenzhen International Graduate School), China; Universities Stable Funding Key Projects (WDZC20200821150704001); The Shenzhen Bay Laboratory, Oncotherapeutics (21310031), China; Supported by 10.13039/501100013290 National Key R & D Program of China (2023YFA0913602 ); the 10.13039/501100001809 National Natural Science Foundation of China (grant no. 82172618 ).

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.tranon.2024.102103.
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