MCL1 regulates cell death, tumor growth and chemosensitivity to sabutoclax in ovarian adenocarcinoma 

Received: 19 March 2019 / Accepted: 15 September 2019
Ⓒ Springer-Verlag GmbH Germany, part of Springer Nature 2019

This research was conducted to study the role of MCL1 in ovarian adenocarcinoma cell death and survival as well as chemosensitivity to sabutoclax. Both in vitro and in vivo assays including qRT-PCR, Western blot, CCK-8, caspase 3/7 activa- tion, colony foci formation assay and xenograft assay were conducted. Except for the xenograft assay, the other experiments were conducted at the cellular level and they were carried out to assess cell activities such as viability, programmed death and proliferation. SKOV3 and OVCAR3 cell lines were used as the cell models for all experiments. It was proved that MCL1 was overexpressed in ovarian adenocarcinoma (tissues and cells) at RNA and protein levels. MCL1 knockdown was also discovered to suppress the viability and proliferation of ovarian adenocarcinoma cells in vivo and in vitro. Lastly, it was found that MCL1 knockdown significantly promoted ovarian carcinoma cell death and the sensitivity to sabutoclax. Thus, we concluded that MCL1 acted as a cancer facilitator in ovarian adenocarcinoma and it is also a suppressor of sabutoclax sensitivity.

Keywords MCL1 . Tumor growth . Sabutoclax . Ovarian adenocarcinoma

Introduction Ovarian cancer is the second most common cause of mortality of female reproductive malignancies (Englert-Golon et al. 2017; Jayson et al. 2014; Koshiyama et al. 2017). In China, the incidence rate of ovarian cancer has raised over the past decade. It was reported that over fifty thousand new cases were diagnosed and approximately 20,000 deaths were caused by ovarian cancer in 2015 (Shi et al. 2017). New therapeutic approaches have been sought to tackle these issues. Nevertheless, the ovarian cancer–related high mortality rate has remained constant. Maximal surgery cytoreduction repre- sents the current standard of care in treating ovarian cancer (Correa et al. 2017; Polom et al. 2016) and is most commonly followed by platinum-based chemotherapy (Whicker et al. 2016). Despite the surgery representing the standard treatment and allowing for a good initial response, some nuances com- promise the healing process. Common problems include lack
of early-stage symptoms leading to late diagnosis, poor re- sponse to treatment owing to late diagnosis, post-therapy re- gression and need for further treatment, resistance to platinum-based chemotherapy (acquired chemoresistance causes relapse and leads to low survival rates of about 30%) and most importantly, lack of funds for treatment by some patients (Arnold et al. 2005; Bonnefond et al. 2015; Denoyelle et al. 2014; Whicker et al. 2016).
While cancer-causative factors exist, other factors are pro- liferative of the tumor cell. One such factor is apoptosis-relat- ed. Failure to initiate apoptosis promotes the development of tumors and prevents cancer therapy–induced cell death (Campbell et al. 2018). The mitochondria-related apoptotic property is monitored and regulated by the BCL-2 family. Within the BCL-2 family, some trigger programmed cell death, while others are anti-apoptotic. MCL1 is one of the latter group members of the BCL-2 family, interfering with mitochondrial events that instigate the release of an apoptosis

initiation protein, cytochrome c. MCL1 is primarily located on
the outer mitochondrial membrane and some intracellular* Cui Li
[email protected]

1 Department of Gynaecology and Obstetrics, Yantai Affiliated Hospital, Binzhou Medical College, No. 717 Jinbu Street, Muping District, Yantai 264100, Shandong, China

membranes. The protein is supposedly amplified in human non-small cell lung cancer (Allen et al. 2011), in advanced human prostate cancer and in other carcinomas and leukemia (Jackson et al. 2012) where its overexpression correlates with poor patient prognosis and survival (Allen et al. 2011).The overexpression of the anti-apoptotic protein members of the BCL-2 family is a major proliferative factor of ovarian cancer cells. MCL1 is one of those serving as a gateway pro- tein that guards against apoptosis in ovarian carcinoma (Bonnefond et al. 2015; Denoyelle et al. 2014). Its overexpres- sion in ovarian carcinoma is linked to poor prognosis (Bonnefond et al. 2015; Habata et al. 2016; Sugio et al. 2014; Wang et al. 2017). If inhibited, apoptosis is triggered in chemoresistant ovarian cancer cells. Regulatory studies have revealed that the downregulation of MCL1 is markedly effective (Denoyelle et al. 2014).

Recently, the creation of novel inhibitors of the anti- apoptotic members of the BCL-2 family has seen tremendous progress. These inhibitors can reinstate apoptosis of cancer cells. BH3-mimetics, small molecules that function by mim- icking BH3-only proteins (effectors of mitochondria- mediated cell apoptosis, such as BAX and BAK) and freeing pro-apoptotic members of the BCL-2 family, are very prom- ising drugs for cancer treatments (Campbell et al. 2018; Dai et al. 2016; Opydo-Chanek et al. 2017). As good as other BH3-mimetics, especially ABT-737 and ABT-263, have been in preclinical and clinical trials in some hematological malig- nancies, they have no binding ability to MCL1 (Varadarajan et al. 2013) and, therefore, cannot reverse its anti-apoptotic property and initiate cancer cell death. There is a need for a therapeutic option capable of offsetting the acquired chemoresistance induced by the overexpression of MCL1. Sabutoclax is a BH3-mimetic and it is an optically pure apogossypol derivative. The in vitro and in vivo efficacy of sabutoclax has been improved and it has been reported to inhibit tumorigenesis in prostate cancer (Dash et al. 2011; Jackson et al. 2012; Sarkar et al. 2016; Varadarajan et al. 2013). However, its usefulness in ovarian carcinoma has not been tested before. This research is the first of its kind to investigate the role of MCL1 in ovarian adenocarcinoma, par- ticularly in cell growth, apoptosis and chemosensitivity to sabutoclax. The goal is to shed new light on the mechanism of ovarian adenocarcinoma chemoresistance to sabutoclax, the comprehension of which would contribute to the develop- ment of an ovarian adenocarcinoma treatment method.
Methods and materials Tissue samples and cell lines

The tissue samples were obtained from patients who were diagnosed with ovarian adenocarcinoma during June 2016– October 2017. All the patients signed an informed consent and the ethics committee of the Yantai Affiliated Hospital, Binzhou Medical College, approved this research. The im- mortalized ovarian epithelial cell line H8 was purchased from BNCC and cultured in 50% RPMI-1640+40% FBS. The used
ovarian adenocarcinoma cell lines included Ect1/E6E7 (BNCC, 50% EMEM+40%F BS), A2780, OVCAR3 (ATCC, 80% RPMI-1640+20% FBS), SKOV3 (ATCC, 90%
McCoy’s 5a Medium Modified +10%FBS) and Caov3 (ATCC, 90% DMEM+10% FBS).siRNA construction and cell transfectionCells were cultured in corresponding cell culture. They were trypsinized, counted and reseeded 12–16 h before siRNA knockdown (cells typically reached ~ 50% confluence). siRNA (designed and synthesized by GenePharma, Shanghai, China; sequences are shown in Table 1) was suspended in 1× siRNA buffer to reach a final concentration of 5 μM. Then 5 μl of the previous solution was mixed with serum-free medium properly. A 1-μl volume of DharmaFECT 1 transfection reagent (cat no. T-2001-03, Dharmacon/Thermo Scientific, Waltham, MA, USA) was properly mixed with 99 μl of serum-free medium. The siRNA solution was added to the DharmaFECT solution and mixed well. A total of 600 μl corresponding complete medium was mixed with the resulting mixture to make the final concentration of siRNA solution 25 nM. The original culture media were removed and the final siRNA solution was added to the cells and incubated for 1 day before mRNA analysis and 2 days before protein analysis. Experiments were performed in triplicate as a minimum.qRT-PCRRNA was extracted from tissues and cells using QIAZOL reagent (Qiagen, Shanghai, China). cDNA was synthesized from 2 μg extracted RNA using random primers and Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Fisher Scientific, Shanghai, China). SYBR Green PowerUp SYBR Green Master Mix was used in this experi- ment, which was performed using a 7900HT Fast RT-PCR thermocycler (Applied Biosystems, MA, USA). The primers for our PCR workflow are as follows: 5 ′-GAGT CAACGGATTTGGTCGT-3′ (forward) and 5′-TTGA TTTTGGAGGGATCTCG-3′ (reverse) for GAPDH and 5′- GGGCAGGATTGTGACTCTCATT-3′ (forward) and 5′- GATGCAGCTTTCTTGGTTTATGG-3′ (reverse). Both sets of primers were validated using the standard PCR method. GAPDH was chosen to be the reference gene. The data were analyzed using the Ct method.Western blotTissues were washed in PBS and lysed with RIPA (formula referred to in Vershinin et al. 2016). Cell lysates were separat- ed by SDS-PAGE and the electro-separated proteins were transferred to a polyvinylidene difluoride membrane. The

Table 1 siRNAs for MCL1 gene
that were used in this study No. Sense 5′–3′ Anti-sense 3′–5′ GC% Position
GGAGAUGUCUAUAAA 42 8903–8925 3 GGUGGAGA UUUGAGAAUAA CCACCUCUAAACUCUUAUU 37 6942–6964 transferred proteins were then incubated with anti-LC3 anti- body (cat no. ab51520, Abcam, Cambridge, UK). Anti-rabbit antibody conjugated with horseradish peroxidase was used as secondary antibody and the membrane was visualized using a chemiluminescent substrate (ImmunoStar LD; Wako Pure Chemical Industries, Ltd.). In terms of protein amount deter- mination, bicinchoninic acid (BCA) protein assay was con- ducted using a BCA kit (Pierce Chemical Co.) on the super- natants of the cell lysates. CCK-8 Cells were plated in a 96-well plate in triplicate at a density of approximately 2000 cells per well. Cell viability was deter- mined using CCK-8 reagent (Dojindo Laboratories, Kumamoto, Japan).

Colony formation experiment The cells were incubated in 4 ml 0.25% trypsin at 37

°C for 3–5 min until the cells appeared round. Cells were then suspended in 10-ml media with 10% FBS. The cell number was counted using a hemocytometer. A total of 500 cells were seeded into every 6-cm dish. After continuous culture for approximately 2 weeks un- til cells in control plates formed colonies with a reasonable size (approximately 50 cells per colony), the media were abandoned and cells were washed with PBS buffer. A volume of 2–3 ml fixation solution (4% paraformaldehyde) was incubated with cells for 5 min at room temperature. After that, 0.1% crystal violet solu- tion was incubated with cells at room temperature for 60 min. The cells were detached by pipetting in 10-ml media with 10% FBS. The crystal violet was carefully washed off with tap water. The dishes were air-dried on a tablecloth at room temperature for 1–3 days. The number of colonies was counted under a stereomicro- scope (Nikon Eclipse TS100, Tokyo, Japan).

Caspase 3/7 activation Cells were plated in triplicate in a 96-well plate at a density of 2000 cells per well. Caspase activation was detected using the Caspase-Glo 3/7 Assay system (Promega, Shanghai, China).Flow cytometry assay to test cell apoptosis The cells were fixed in 75% cold ethanol for 70 min, then washed in PBS buffer twice. The cells were then centrifuged at 850g for 5 min and the supernatant was removed. After that, the cells were treated with ribonuclease. Thereafter, 50 μl RNase (100 μg/ml) was added to the cells to ensure that only

 1 MCL1 gene expression is upregulated in ovarian adenocarcinoma tissues and cell lines.a MCL1 mRNA expression in non-malignant tissue is significantly lower than that in cancerous tissue. **P < 0.05, comparing with cancerous tissue. b MCL1 mRNA expression in non-malignant cell lines H8 and E6E7 was significantly lower than that in the four cancerous
cell lines. ***P < 0.01, comparing with mRNA level in H8 cell line. c MCL1 protein expression in non-malignant cell line was significantly lower than that in the four ovarian carcinoma cell lines. **P < 0.05, comparing with protein level in H8 cell line

 2 MCL1 supports cell population growth and clonogenic survival as well as inhibits apoptosis of ovarian adenocarcinoma cells. a In both SKOV3 and OVCAR3 cell lines, the MCL1 mRNA level is

significantly higher in the NT (non-transfected) group than in MCL1 siRNA no. 1–3 groups. **P < 0.01 and ***P < 0.001, compared with the NT group. b In both SKOV3 and OVCAR3 cell lines, the MCL1 protein level is significantly higher in the NT group than in MCL1 siRNA no. 1–3 groups. **P < 0.01 and ***P < 0.001, compared with the NT group. c, d The cell population growth determined by CCK-8 assay of SKOV3 cells (c) and OVCAR3 cells (d) in the NT group is significantly higher than that in no. 1–3 groups. *P < 0.05, compared with the NT group. e, f Colony formation assay results show that colony formation in the NT group is significantly stronger than that in no. 1–3 groups in SKOV3 (e, e′, e′′, e′′′) and OVCAR3 (f, f’′, f′′, f′′′) cells. g, h The quan- titative analysis of e–f′′′. ****P < 0.001, compared with the NT group. i, j Caspase 3/7 activation assay results show that caspase 3/7 activation in the NT group is significantly weaker than that in no. 1–3 groups. *P <
0.05 and ***P < 0.001, compared with the NT group

DNA were stained. A volume of 400 μl of 50 μg/ml propidium iodide ( PI) and Annexin V- FITC ( BD Pharmingen, San Jose, CA, USA) were incubated with cells at 37 °C for 40 min. Lastly, the cell apoptosis rate was mea- sured using a flow cytometer (FACS Calibur, BD Biosciences, San Jose, CA, USA).Xenograft tumor growth assay

All animal experiments with cell line xenografts complied with the guidelines for the care and use of laboratory animals of the hospital. Female BALB/c nude mice were purchased from the Beijing Vital River Laboratory Animal Technology Center (Beijing, China) and kept in a temperature-controlled, sterile environment with 12-h light and dark cycles. All procedures were conducted on 4–6-week-old mice. Mice were randomized into groups (blank control group and sabutoclax treatment group). In the blank control group, cells transfected within PBS:matrigel (1:1 ratio) mixture were injected into the mice. In the treatment group, 2 mg/kg sabutoclax was dosed daily. Both SKOV3 and OVCAR3 cell lines were used to build the xenograft tumor models. A total of 5 × 107 cells/ml were trypsinized with 1× trypsin solution. The detached cells were collected and spun at 300g for 3 min. The supernatant was removed and the sediment was suspended within 2-ml culture media. The suspended solution was then diluted to reach a concentration of 5 × 106 cells/ml. The mixture was then injected into the left side of the dorsal flank of each mouse subcutaneously at an inoculation depth of 1 cm. The tumor size was mea- sured every week using a caliper and the final size was determined 30 days after the injection. The volume of a tumor (cm3) was calculated as 1/2(L × W2), in which L and W stands for the length and width of the tumor in centimeters, respectively.

Statistical analysis Statistical analysis was conducted using GraphPad Prism ver.

6. A t test was used for pair data whereas ANOVA test was used for multiple data in this study. Each experiment had 3 parallels to exclude the randomness. Data were presented in a form of average ± standard deviation. P < 0.05 was considered as the threshold of statistical significance.


MCL1 gene expression in ovarian adenocarcinoma tissues and cell lines

Firstly, to determine the expression level of MCL1, MCL1 mRNA and protein expression was measured in tissues and cell lines of ovarian adenocarcinoma using PCR and WB. The results revealed that the MCL1 level was significantly higher (more than 2-fold) in ovarian adenocarcinoma tissues ( 1a) and cell lines ( 1b, c) than in non-malignant tissues and cell line H8. SKOV3 and OVCAR3 cell lines were chosen for further analysis.MCL1 knockdown retarded cell viability

and clonogenic survival but promoted apoptosis of ovarian adenocarcinoma cells by siRNA We conducted CCK-8, colony foci formation and caspase 3/7 activation assays to reveal the role of MCL1 in cell viability, growth and death in SKOV3 and OVCAR3 cell lines. We firstly confirmed that the knockdown of MCL1 by siRNA was successful at both mRNA and protein levels using PCR and WB (. 2a, b). Then the cell viability and growth abil- ities were studied using CCK-8 and colony formation assay, the results of which showed that the knockdown of MCL1 significantly reduced cell viability (. 2c, d) and growth ( 2e–h) of both SKOV3 and OVCAR3 cell lines. Similarly, caspase 3/7 activation was studied and the results suggested that MCL1 knockdown led to increased cell apo- ptosis (. 2i, j). The three siRNAs against MCL1 gene were all effective in reducing MCL1 gene expression. siRNA no. 3 showed the highest efficiency, thus were chosen for further experiments.

MCL1 knockdown reduces cell growth and tumor growth in xenograft models of ovarian carcinoma by pan-BCL-2 inhibitor sabutoclax (IC50, 0.20 μM)

When exposed to sabutoclax, cancer cell lines SKOV3 and OVCAR3 with MCL1 knockdown showed significantly re- duced cell viability compared with the immortalized ovarian epithelial cell line H8 with MCL1 knockdown (. 3a). We

. 3 MCL1 inhibition impairs cell growth and tumor growth in in vivo xenograft models of ovarian adenocarcinoma. a The cell surviving (viability) outcomes: H8 cell line with MCL1 gene knockdown is statis-

tically indifferent to the change of sabutoclax concentrations; SKOV3 and OVCAR3 cells (with MCL1 knockdown), on the other hand, show significantly reduced cell survival compared with H8 cells with MCL1 gene knockdown. *P < 0.05, compared with H8 cells. b Both SKOV3 and OVCAR3 cell lines (all with MCL1 knockdown) demonstrate signif- icantly increased caspase 3/7 activation when exposed to sabutoclax. *P
< 0.05, compared with the control group, in which no sabutoclax or other drugs were used. c, d The number of colonies of SKOV3 (c) and OVCAR3 (d) cells (all with MCL1 knockdown) is significantly reduced when exposed to sabutoclax. *P < 0.05, compared with the control group. e, f Cell apoptosis rate of SKOV3 (e) and OVCAR3 (f) cells (all with MCL1 knockdown) is significantly higher when exposed to sabutoclax compared with the control group. *P < 0.05, compared with the control group. g, h When treated with sabutoclax, the mice injected with either SKOV3 cell line (g) or OVCAR3 cell line (h) (all with MCL1 knock- down) demonstrate significantly reduced tumor volumes compared with those injected with parental SKOV3 and OVCAR3 cells. *P < 0.05, compared with the control group, in which the MCL1 of cancer cells was not silenced

then determined that the IC50 of sabutoclax for ovarian cancer cell lines SKOV3 and OVCAR3 was 0.20 μM. We subse- quently detected caspase 3/7 activation in SKOV3 and OVCAR3 cell lines with MCL1 knockdown. Caspase 3/7 ac- tivation is an apoptotic molecular marker. Our experimental results showed that in either SKOV3-siMCL1 cells or OVCAR3-siMCL1 cells, the addition of sabutoclax led to a significantly enhanced level of caspase 3/7 activation (en- hanced by more than 1.5-fold in the SKOV3 cell line and by approximately 2.5-fold in the OVCAR3 cell line, . 3b), suggesting that the forced loss of function of the MCL1 gene in ovarian cancer cells could induce cell apoptosis. Then, we conducted colony foci formation assay to determine the influ- ence of the knockdown of MCL1 gene on the proliferation of ovarian cancer cells. The results showed that in both SKOV3 and OVCAR3 cell lines with MCL1 knockdown, the addition of sabutoclax led to significantly reduced colony numbers (by more than half) ( 3c, d). Also, flow cytometric assay was conducted to determine the cell apoptosis. Again, the addition of sabutoclax caused significantly increased cell apoptosis (by more than 3-fold) in both cell lines with loss of MCL1 gene (. 3e, f). In vivo experiments were done to confirm the in vitro results. We found that MCL1 gene absence plus expo- sure to sabutoclax significantly reduced tumor volume forma- tion (. 3g, h). Taking together, our results suggest that MCL1 gene knockdown could reduce ovarian cancer cell pro- liferation and tumor growth in xenograft models of ovarian carcinoma exposed to pan-BCL-2 inhibitor sabutoclax.

Discussion Our studies show that the MCL1 gene expression is upregu- lated in tissues and cell lines of ovarian adenocarcinoma. The

overexpression of this protein parallels cancer cell population growth, which suggests that MCL1 promotes cancer cell pro- liferation and clonogenic survival, as well as inhibits the apo- ptosis of ovarian adenocarcinoma cells. However, the use of sabutoclax on in vivo xenograft models of ovarian adenocar- cinoma (pan-BCL-2 inhibitor sabutoclax experiments) knocked down MCL1, suppressing its anti-apoptotic feature. Downregulating MCL1 also impaired tumor growth and inhibited the growth of ovarian adenocarcinoma models in vitro and in vivo. These findings are in line with recent studies that demonstrated the efficacy of sabutoclax in vivo and in vitro when treating other cancer forms (Jackson et al. 2012). Jackson et al., in their investigation of the impact of sabutoclax on in vivo and in vitro xenograft and transgenic mouse models of prostate cancer, found the antagonists of pan-active BCL-2 proteins to mediate apoptosis in castrate- resistant prostate cancer cells of Tgfbr2ColTKA mice and hu- man subcutaneous, orthotopic and intratibial xenograft pros- tate cancer models by blocking c-Met activation. Sabutoclax could, therefore, equally halt MCL1 overexpression and inhib- it its anti-apoptotic property in ovarian adenocarcinoma.
Given its relative novelty in laboratory test treatments of cancerous cells, the exact mechanism of action of sabutoclax is yet elusive, although it functions by mimicking BH3-only proteins and freeing pro-apoptotic BCL-2 proteins (Campbell et al. 2018). Nevertheless, the drug and other BH3-mimetics have significant implications for the growth and proliferation of cancerous cells. Knocking down or inhibiting MCL1 in vivo and in vitro with various BH3-mimetics highlights how strongly cancer cells depend on the anti-apoptotic protein for proliferation. MCL1 knockdown in ovarian adenocarcino- ma in our experiment culminated in cell death and suppressed tumor growth. Other investigations show similar characteris- tics. In vivo and in vitro experiments on genetically engineered mouse models in previous research revealed that inhibiting MCL1 overexpression slowed breast cancer pro- gression, with resistance to chemotherapy potentially not be- ing acquired in response to therapy but rather due to the high presence of MCL1 in cancerous cells (Campbell et al. 2018). Merino et al., on their part, argued that knocking down MCL1 increases chemotherapy efficacy by re-sensitizing resistant tu- mors to therapy (Merino et al. 2017), a fact supported in a 2012 in vivo investigation in which knocking down MCL1 mRNA and protein levels substantially impeded its upregula- tion and apoptosis inhibition by rapidly phosphorylating ERK1/2 and Elk-1 transcription factor and re-sensitizing ovar- ian cancer cells to chemotherapy (Goncharenko-Khaider et al. 2012). Another in vitro investigation on ovarian cancer cells revealed that downregulating BAG3 had an indirect inhibitory effect on the expression of MCL1, altering ovarian cancer cell proliferation, and re-sensitizing tumor cells to chemotherapy (Habata et al. 2016). The significant implications in downreg- ulating MCL1 in ovarian adenocarcinoma, therefore, point to the arrest of proliferation and re-sensitization of tumor cells to chemotherapy.

Sabutoclax has previously been shown to inhibit the expression of MCL1 in a variety of cancers. However, the product is yet to see the clinical phase of trials, with most investigations conducted in vivo and in vitro, as well as on xenograft mouse models. Being the first to use the apogossypol derivative on ovarian adenocarcino- ma in vivo and in vitro means there is no previous record to back the impact of sabutoclax on ovarian can- cer cells or adenocarcinoma. Nevertheless, other in vitro and in vivo treatments of carcinogen-induced carcino- mas, such as oral squamous cell carcinoma cells, with sabutoclax reportedly knocked down MCL1 expression, induced cancer-specific cell death via apoptosis and tox- ic mitophagy and significantly decreased tumor growth (Maji et al. 2015). This parallel finding would suggest that carcinogen-induced ovarian cancer would undoubt- edly be sensitive to the apoptosis-inducing property of sabutoclax, which acts by inhibiting or downregulating the expression of MCL1. Possessing features found in other cancer treatment drugs while being able to regu- late MCL1 directly gives sabutoclax an added advantage as demands for its use in clinical trials intensify.
Given the inadequacy in the types of drugs that can directly downregulate MCL1 expression in cancer cells, finding fur- ther treatment options is vital. MCL1 has an extremely short half-life owing to ubiquitin-mediated proteasomal degradation when compared with other BCL-2 proteins (Thomas et al. 2013), which gives an added incentive to provide a therapeutic solution to MCL1 overexpression. We have shown in our studies, for the first time, that sabutoclax can suppress MCL1 overexpression, which triggers the anti-proliferation of ovarian adenocarcinoma, enables chemosensitivity in tu- mor cells and reduces tumor size. The drug, therefore, through its properties, could be of high therapeutic value in the treat- ment of ovarian adenocarcinoma and other cancer cells going forward.

Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest.

Ethical approval The ethics committee of the Yantai Affiliated Hospital, Binzhou Medical College, approved this research.


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