• Toxicological Research
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• v.35 no.2
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• pp.167-179
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• 2019

Ovarian cancer is the fifth main cause of pre-senescent death in women. Although chemotherapy is generally an efficient treatment, its side effects and the occurrence of chemotherapeutic resistance have prompted the need for alternative treatments. In this study, ${\alpha}$-mangostin and apigenin were evaluated as possible anticancer alternatives to the chemotherapeutic drug doxorubicin, used herein as a positive control. The ovarian adenocarcinoma cell line SKOV-3 (ATCC No. HTB77) was used as model ovarian cancer cells, whereas the skin fibroblast line CCD-986Sk (ATCC No. CRL-1947) and lung fibroblast line WI-38 (ATCC No. CCL-75) were used as model untransformed cells. Apigenin and doxorubicin inhibited the growth of SKOV-3 cells in a dose- and time-dependent manner. After 72 hr exposure, doxorubicin was mostly toxic to SKOV-3 cells, whereas apigenin was toxic to SKOV-3 cells but not CCD-986Sk and WI-38 cells. ${\alpha}$-Mangostin was more toxic to SKOV-3 cells than to CCD-986Sk cells. A lower cell density, cell shrinkage, and more unattached (floating round) cells were observed in all treated SKOV-3 cells, but the greatest effects were observed with ${\alpha}$-mangostin. With regard to programmed cell death, apigenin caused early apoptosis within 24 hr, whereas ${\alpha}$-mangostin and doxorubicin caused late apoptosis and necrosis after 72 hr of exposure. Caspase-3 activity was significantly increased in ${\alpha}$-mangostin-treated SKOV-3 cells after 12 hr of exposure, whereas only caspase-9 activity was significantly increased in apigenin-treated SKOV-3 cells at 24 hr. Both ${\alpha}$-mangostin and apigenin arrested the cell cycle at the $G_2/M$ phase, but after 24 and 48 hr, respectively. Significant upregulation of BCL2 (apoptosis-associated gene) and COX2 (inflammation-associated gene) transcripts was observed in apigenin- and ${\alpha}$-mangostin-treated SKOV-3 cells, respectively. ${\alpha}$-Mangostin and apigenin are therefore alternative options for SKOV-3 cell inhibition, with apigenin causing rapid early apoptosis related to the intrinsic apoptotic pathway, and ${\alpha}$-mangostin likely being involved with inflammation.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.6
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• pp.2453-2458
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• 2015

Background: X-linked inhibitor of apoptosis protein (XIAP) associated factor 1 (XAF1) exhibits aberrantly low or absent expression in various human malignancies, closely associated with anti-apoptosis and overgrowth of cancer cells. However, limited attention has been directed towards the contribution of XAF1 to invasion, apoptosis, and cisplatin (DDP)-resistance of epithelial ovarian cancer (EOC) cells. This study aimed to evaluate the potential effects of XAF1 on invasion, cell cycle, apoptosis, and cisplatin-resistance by overexpressing XAF1 in SKOV-3 and SKOV-3/DDP cells. Methods and Results: The pEGFP-C1-XAF1 plasmid was transfected into SKOV-3 and SKOV-3/DDP cells, and the expression of XAF1 at both mRNA and protein levels was analyzed by reverse transcription-PCR and Western blotting. Overexpression of XAF1 suppressed XIAP expression in both SKOV-3 and SKOV-3/DDP cells. Transwell invasion assays demonstrated that XAF1 exerted a strong anti-invasive effect in XAF1-overexpressing cells. Moreover, flow cytometry analysis revealed that XAF1 overexpression arrested the cell cycle at G0/G1 phase, and cell apoptosis analysis showed that overexpression of XAF1 enhanced apoptosis of SKOV-3 and SKOV-3/DDP cells apparently by activating caspase-9 and caspase-3. Furthermore, MTT assay confirmed a dose-dependent inhibitory effect of cisplatin in the tested tumor cells, and overexpression of XAF1 increased the sensitivity of SKOV-3 and SKOV-3/DDP cells to cisplatin-mediated antiproliferative effects. Conclusions: In summary, our data indicated that overexpression of XAF1 could suppress XIAP expression, inhibit invasion, arrest cell cycle, promote apoptosis, and confer cisplatin-sensitivity in SKOV-3 and SKOV-3/DDP cells. Therefore, XAF1 may be further assessed as a potential target for the treatment of both cisplatin-resistant and non-resistant EOCs.

• Journal of Life Science
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• v.33 no.2
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• pp.129-137
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• 2023

Reactive oxygen species (ROS) play an essential role in a variety of cellular physiological phenomena. The present study assessed the signaling axis that mediates the lysophosphatidic acid (LPA)-induced migration of SKOV-3 cells. Insulin-like growth factor-1 (IGF-1) stimulated SKOV-3 cell migration in a time- and dose-dependent manner. Similarly, LPA stimulated SKOV-3 cell migration and the phosphorylation of Akt in a time- and dose-dependent manner. The pharmacological inhibition of LPA receptors (LPA₁/LPA₃) significantly suppressed LPA-induced SKOV-3 cell migration. However, IGF-1-induced SKOV-3 cell migration was not affected by the inhibition of LPA1 and LPA3. Pharmacological inhibition of phosphoinositide 3-kinase (PI3K) or Rho-associated kinase (ROCK) significantly suppressed LPA-induced migration, whereas the inhibition of MAPK kinase (MEK) had no effect. Inhibition of PI3K or ROCK completely suppressed LPA-induced ROS generation, and suppression of nicotinamide adenine dinucleotide phosphate oxidase (NOX) or chelation of ROS by N-acetylcysteine (NAC) blocked LPA-induced SKOV-3 cell migration. LPA-induced ROS generation was suppressed by silencing Rictor or Akt1 but not Raptor or Akt2. Silencing Rictor or Akt1 significantly suppressed LPA-induced SKOV-3 cell migration, whereas silencing Raptor or Akt2 had no effect. Finally, the overexpression of the constitutively active form Akt1 (CA-Akt1) significantly enhanced the LPA-induced migration of SKOV-3 cells. Given these results, we suggest that LPA stimulates SKOV-3 cell migration by ROS generation, which is mediated by the mTORC2/Akt1/NOX signaling axis.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.12
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• pp.7197-7201
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• 2013

Aim: To investigate the effects of diallyl trisulfide (DT) on apoptosis of cisplatin (DDP)-resistant human epithelial ovarian cancer SKOV-3 cells (SKOV-3/DDP), and the role of p53 upregulated modulator of apoptosis (PUMA). Methods: SKOV-3/DDP cells were randomly divided into control, DT, DPP and DPP+DT groups, which were treated with DT or combined DT and DDP. All cells were incubated for 48 h. and apoptosis rates were assessed by flow cytometry. mRNA and protein expression of PUMA, Bax and Bcl-2 was determined by RT-PCR and Western blot assays, respectively. Results: Compared with control group, the apoptosis rates of SKOV-3/DDP cells in DT groups were obviously increased, with dose-dependence (P < 0.05), the mRNA and protein expressions of PUMA, Bax also being up-regulated (P < 0.05), while those of Bcl-2 were down-regulated (P < 0.05). Compared with DT groups, the apoptosis rate in the DDP+DT group was significantly increased (P < 0.05). After knockdown of PUMA with specific siRNA, the apoptosis rate of SKOV-3/DDP cells was obviously decreased (P < 0.05). Conclusion: DT can promote the apoptosis of SKOV-3/DDP cells with PUMA playing a critical role.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.20
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• pp.8595-8601
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• 2014

Background: miR-200a expression is frequently altered in numerous cancers. The aim of the present study was to determine the role of microRNA-200a in advanced ovarian carcinomas. Materials and Methods: We measured miR-200a expression in 72 matched normal ovarian tissues and advanced ovarian carcinomas, and also two ovarian carcinoma cell lines (SKOV3 and SKOV3.ip1 - the latter being more invasive and metastatic than the parental SKOV3) by stem-loop real-time RT-PCR based on TaqMan microRNA assay using U6 as a reference. Levels of miR-200a expression were compared by disease stage, tumor grade, histology, and lymph node involvement. To evaluate the role of microRNA-200a, cell proliferation and invasion of SKOV-3 and SKOV-3.ip1 were analyzed with miR-200a inhibitor/mimic transfected cells. Results: Of 72 paired samples, 65 cancer tissues overexpressed microRNA-200a greater than two fold in comparison with matched normal epithelium. Specifically, patients with lymph node metastasis showed significant elevation. The level correlated with clinicopathological features, including high tumor grade, late disease stage, most notably with lymph node metastasis, but not with tumor histology. In addition, SKOV-3.ip1 cells also overexpressed miR-200a compared with SKOV-3, and miR-200a inhibitor transfected SKOV-3.ip1 cells showed significant reduction in cellular proliferation and invasion, while a miR-200a mimic stimulated the opposite behavior. Conclusions: We provide definitive evidence that miR-200a is up-regulated in a significant proportion of advanced ovarian carcinomas, and that elevated miR-200a expression facilitates tumor progression. Our findings support the notion that miR-200a is an onco-microRNA for ovarian cancer, and elevation is a useful potential diagnostic indicator. This study also provides a solid basis for further functional analysis of miR-200a in advanced ovarian cancer.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.2
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• pp.797-802
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• 2014

We screened a small molecular library that was designed and independently synthesized in vitro and found a new drug (MY-03-01) that is active against ovarian cancer. We established that MY-03-01 effectively inhibited SKOV-3 cell survival in a dose-dependent manner, based on cell viability rates, and that it not only induced SKOV-3 apoptosis by itself, but also did so synergistically with paclitaxel. Secondly, when MY-03-01 was applied at $40{\mu}M$, its hemolytic activity was less than 10%, compared with the control, and there was almost no damage to nor mal cells at this concentration. In addition, we used DAPI staining and flow cytometry to show that MY-03-01 could significantly induce apoptosis of SKOV-3 cells. Finally, we found that MY-03-01 likely induced SKOV-3 apoptosis by activating caspase3 and caspase9 through the mitochondrial pathway.

• Journal of Ginseng Research
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• v.44 no.1
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• pp.96-104
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• 2020

Objectives: Oleanolic acid, a minor element of ginsenosides, and its derivatives have been shown to have cytotoxicity against some tumor cells. The impact of cytotoxic effect of oleanolic acid 3-acetate on ovarian cancer SKOV3 cells and endometrial cancer HEC-1A cells were examined both in vivo and in vitro to explore the underlying mechanisms. Methods: Cytotoxic effects of oleanolic acid 3-acetate were assessed by cell viability, phosphatidylserine exposure on the cell surface, mitochondrial release of cytochrome C, nuclear translocation of apoptosis-inducing factor, depolarization of mitochondrial transmembrane potential (∆Ψ_m), and generation of reactive oxygen species (ROS). In vivo inhibition of tumor growth was also assessed with xenografts in immunocompromised mice. Results: Oleanolic acid 3-acetate exhibited potent cytotoxicity toward SKOV3 and HEC-1A cells by decreasing cell viability in a concentration-dependent manner. Importantly, oleanolic acid 3-acetate effectively suppressed the growth of SKOV3 cell tumor xenografts in immunocompromised mice. Furthermore, oleanolic acid 3-acetate induced apoptotic cell death as revealed by loss of ∆Ψ_m, release of cytochrome c, and nuclear translocation of apoptosis-inducing factor with a concomitant activation of many proapoptotic cellular components including poly(ADP-ribose) polymerase, Bcl-2, and caspases-8, caspase-3, and caspase-7. Oleanolic acid 3-acetate, however, caused a decrease in ROS production, suggesting the involvement of an ROS-independent pathway in oleanolic acid 3-acetate-induced apoptosis in SKOV3 and HEC-1A cells. Conclusion: These findings support the notion that oleanolic acid 3-acetate could be used as a potent anticancer supplementary agent against ovarian and endometrial cancer. Oleanolic acid 3-acetate exerts its proapoptotic effects through a rather unique molecular mechanism that involves an unconventional ROS-independent but mitochondria-mediated pathway.

• Korean Journal of Clinical Laboratory Science
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• v.47 no.4
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• pp.298-305
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• 2015

Stem cell-like tumor cells are reported to be the main reason for tumor recurrence and metastasis. As one of the new approaches to overcome cancer, studies are emerging to inhibit the expressions of stem cell transcriptional factors (Oct4, Sox2, Klf-4, and Lin28) in cancer cells. MicroRNAs are master genetic regulators that can control development and differentiation of stem cells. In this study using various ovarian tumors (Skov3, Ovcar3, Tov112D, Tov21G, PA-1 and Hsc832(c)T), we examined the expressions of stem cell-related transcription factors, and the biological changes in cell survival and growth by miR-126 that targets stem cell transcriptional factors. We observed that treatment of miR-126 induced the morphological changes and cell suspension in most cells. In addition, miR-126 induced gradual regression of cell division except Skov3 cells, especially significant time-dependent reduction in Tov112D, Tov21G and PA-1. When we examined the expression of stem cell transcriptional factors, Sox2 was shown to be down-regulated after miR-126. Our results demonstrate that miR-126 treatment can provide the reversible environment to regulate cell division and to induce cell death of ovarian tumors, suggesting the molecular biological clues for clinical usage.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.8
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• pp.3363-3368
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• 2014

Background: Curcumin, a phenolic compound extracted from the rhizomes of Curcuma longa, has shown cytotoxic effects against a variety of cancers. The aim of this study was to identify potential microRNA (miRNA) mediators of the anticancer effects of curcumin in ovarian cancer cells. Materials and Methods: SKOV3 ovarian cancer cells were treated with curcumin ($10-60{\mu}M$) and miR-9 expression, cell proliferation, and apoptosis were assessed. The effects of miR-9 depletion on curcumin-mediated growth suppression were also examined. Phosphorylation of Akt and forkhead box protein O1 (FOXO1) was measured in cells with miR-9 overexpression or curcumin treatment. Results: Curcumin caused a significant and dose-dependent increase of miR-9 expression in SKOV3 cells, while significantly impeding cell proliferation and stimulating apoptosis. Depletion of miR-9 significantly (p<0.05) attenuated the growth-suppressive effects of curcumin on SKOV3 cells, coupled with reduced percentages of apoptotic cells. In contrast, overexpression of miR-9 significantly enhanced the cleavage of caspase-3 and poly(ADP-ribose) polymerase and promoted apoptotic death in SKOV3 cells. Western blot analysis showed that both miR-9 overexpression and curcumin similarly caused a significant (p<0.05) decline in the phosphorylation of Akt and FOXO1, compared to untreated cells. Conclusions: The present study provided evidence that curcumin exerts its cytotoxic effects against SKOV3 ovarian cancer cells largely through upregulation of miR-9 and subsequent modulation of Akt/FOXO1 axis. Further studies are needed to identify direct targets of miR-9 that mediate the anticancer effects of curcumin in ovarian cancer cells.

• Journal of Life Science
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• v.22 no.12
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• pp.1621-1627
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• 2012

Cell motility plays an essential role in many physiological responses, such as development, immune reaction, and angiogenesis. In the present study, we showed that lysophosphatidic acid (LPA) modulates cancer cell migration by regulation of generation of reactive oxygen species (ROS). Stimulation of SKOV-3 ovarian cancer cells with LPA strongly promoted migration. but this migration was completely blocked by pharmacological inhibition of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Inhibition of the ERK pathway had no effect on migration. Stimulation of SKOV-3 ovarian cancer cells with LPA significantly induced the generation of ROS in a time-dependent manner. LPA-induced generation of ROS was significantly blocked by pharmacological inhibition of PI3K or Akt, but inhibition of the ERK signaling pathway had little effect. LPA-induced generation of ROS was blocked by pretreatment of SKOV-3 ovarian cancer cells with an NADPH oxidase inhibitor, whereas inhibition of xanthine oxidase, cyclooxygenase, or mitochondrial respiratory chain complex I had no effect. Scavenging of ROS by N-acetylcysteine completely blocked LPA-induced migration of SKOV-3 ovarian cancer cells. Inhibition of NADPH oxidase blocked LPA-induced migration whereas inhibition of xanthine oxidase, cyclooxygenase, or mitochondrial respiratory chain complex I did not affect LPA-induced migration of SKOV-3 ovarian cancer cells. Given these results, we suggest that LPA induces ROS generation through the PI3K/Akt/NADPH oxidase signaling axis, thereby regulating cancer cell migration.