Corilagin sensitizes epithelial ovarian cancer to chemotherapy by inhibiting Snail­glycolysis pathways.

We identified that corilagin is a major component extracted from a well-known hepatoprotective and antiviral medicinal herb, Phyllanthus niruri L with antitumor activity. Our previous study found that corilagin inhibited the growth of ovarian cancer cells via the TGF-ß/AKT/ERK signaling pathways. Recently, we demonstrated that corilagin enhanced the sensitivity of ovarian cancer cells to chemotherapy. Ovarian cancer cell lines, SKOv3ip, Hey and HO-8910PM-Snail, were treated with different concentrations of corilagin in combination with paclitaxel and carboplatin. Corilagin distinctly enhanced the inhibitory effects of paclitaxel and carboplatin. To understand the mechanisms involved in the chemo-sensitization by corilagin, we performed reverse phase protein array analysis to determine the signaling networks induced by corilagin. We observed that both paclitaxel and carboplatin upregulated the expression levels of several apoptotic and death-related proteins, such as caspase 3, caspase 7 and PDCD4, which were further enhanced when combined with corilagin. Meanwhile, corilagin induced distinct pathways to paclitaxel and carboplatin treatment. We also performed isobaric tags for relative and absolute quantitation proteomics analysis in corilagen-treated ovarian cancer cells. This analysis indicated that corilagin is mainly involved in the glycolysis pathway. Seahorse XF96 extracellular acidification rate analysis confirmed that corilagin inhibited glycolysis by downregulation of CD44 and STAT3. In summary, our observations indicate that corilagin sensitized epithelial ovarian cancer cells to paclitaxel and carboplatin treatment by primarily inhibiting Snail-glycolysis pathways. Corilagin is a herbal medicine with low toxic effects to normal cells, particularly hepatoprotective, and may be an ideal complimentary medicine when combined with highly toxic chemotherapeutic agents.

Intraoperative nerve staining in nerve-sparing radical hysterectomy: a pilot study.

OBJECTIVE: To evaluate the feasibility and effectiveness of intraoperative nerve staining by modified leucomethylene blue (MLB). METHODS: Animal experiment was performed to assure whether the tissues dyed blue by MLB were nerves with microscopic examination. Ten patients with cervical cancer were performed by nerve-sparing radical hysterectomy (NSRH) and nerve staining intraoperatively by MLB. The status of staining was recorded. The post-void residual urine volume after removing was measured by ultrasound. The time to post-void residual urine volume of less than 100 ml and the first defecation were recorded. RESULTS: In animal experiment, the tissues dyed blue obviously showed abundant nerve fibers by microscopic examination. The minor nerves were dyed blue clearly in NSRH. The time to post-void residual urine volume of less than 100 ml after removal of the urethral catheter was 10.3 (7-13) days by records. The time to the first defecation was 67.7 (60-82) h. CONCLUSION: Intraoperative nerve staining by MLB provided a new method for nerve location in NSRH. It was safe, effective and convenient.

Rab5a­mediated autophagy regulates the phenotype and behavior of vascular smooth muscle cells.

Rab5a, a key member of the Rab family of GTPases, was determined to be a regulator of vascular smooth muscle cell (VSMC) proliferation and migration. However, the exact regulatory mechanism remains unclear. As Rab5a has been shown to be associated with autophagy, which is essential for the conversion of VSMCs from a contractile to a synthetic phenotype in order to prevent cell death due to oxidative stress. The present study hypothesized that autophagy may be responsible for the proliferation and migration of VSMCs via the Rab5a protein. The aim of the present study was to evaluate the effect of Rab5a on autophagy in VSMCs. The human aorta vascular smooth muscle cell line, T/G HA­VSMCs, was treated with small interfering (si)RNA against Rab5a and/or platelet­derived growth factor (PDGF). Following treatment, the phenotype transition of the VSMCs was evaluated by detecting the mRNA and protien expression levels of VSMC molecular markers using reverse transcription­quantitative polymerase chain reaction and western blotting, respectively. In addition, autophagy in VSMCs was evaluated by western blotting for autophagy­associated proteins, flow cytometry of acidic vesicular organelles, punctate fluorescence of microtubule associated protein light chain 3 and transmission electron microscopy of typical scattered double­membrane vacuolar structures. Additionally, the proliferation, migration, cell cycle and apoptotic response of VSMCs were detected by sulforhodamine B assay, transwell assay and flow cytometry, respectively. The results revealed that transfection with siRNA against Rab5a led to a significant decrease in Rab5a protein expression, while the reduced expression trend of Rab5a was rescued by intervention with PDGF. Furthermore, cells transfected with siRNA against Rab5a inhibited the autophagy of VSMCs. Downregulated Rab5a inhibited the phenotype transition of VSMCs. Additionally, downregulated Rab5a led to slowed cell growth, decreased numbers of migrated cells, decreased numbers of cells at the G0­G1 phase and a higher apoptosis rate. However, PDGF significantly rescued these phenomena caused by siRNA against Rab5a. These results indicated that Rab5a­mediated autophagy may regulate the phenotype transition and cell behavior of VSMCs through the activation of the extracellular­regulated kinase 1/2 signaling pathway.

Effects of Treg cells and IDO on human epithelial ovarian cancer cells under hypoxic conditions.

The aim of the present study was to explore the effect of hypoxia on ovarian cancer. A total of 6 samples were analyzed: SKOV3­IP cells (ovarian cancer cell line); SKOV3­IP and regulatory T (Treg) cells; SKOV3­IP and cytotoxic T lymphocytes (CTLs); SKOV3­IP and natural killer (NK) cells; SKOV3­IP co-cultured with CTLs and Treg cells; and SKOV3­IP co-cultured with Treg cells and NK cells. The expression of indoleamine 2,3­dioxygenase (IDO) was detected by reverse transcription-polymerase chain reaction (RT­PCR) and western blot analysis. An enzyme­linked immunosorbent assay (ELISA) was used to detect the concentration of transforming growth factor­ß (TGF­ß), interferon­Î³ (IFN­Î³), interleukin­2 (IL­2), interleukin­10 (IL­10), and perforin. Moreover, ovarian cancer cell apoptosis and invasive ability were examined using flow cytometry and a Transwell chamber assay. IDO expression was significantly reduced in hypoxia and enhanced by Treg cells. Treg cells inhibited the IL­2, IFN­Î³ and perforin secretion, and significantly (P<0.05) increased the IL­10 and TGF­ß levels. The effects of Treg cells were enhanced with prolongation of the cell exposure to hypoxic conditions. In addition, Treg cells attenuated the promotive effect of CTLs and NK cells on cancer cell apoptosis. In addition, Treg cells significantly increased the SKOV3­IP invasive ability (P=0.00109) under hypoxic conditions. Our results suggest that IDO and Treg cells may serve as important therapeutic targets for patients with ovarian cancer.

MiR-302c inhibits tumor growth of hepatocellular carcinoma by suppressing the endothelial-mesenchymal transition of endothelial cells.

Endothelial cells (ECs) are critical for angiogenesis, and microRNA plays important roles in this process. In this study, we investigated the function and mechanism of miR-302c in the process of endothelial-mesenchymal transition (EndMT) in ECs. When miR-302c was overexpressed in HUVECs, the motility of the HUVECs was weakened; the expression levels of EndMT markers were also changed: vascular endothelial (VE)-cadherin was up-regulated, whereas ß-catenin, FSP1, and α-SMA were down-regulated. Further in vivo and in vitro experiments showed that the growth of HCC was inhibited when co-cultured or co-injected with HUVECs overexpressing miR-302c. On the contrary, when miR-302c was suppressed in HUVECs, the opposite results were observed. Reporter assays showed that miR-302c inhibited metadherin (MTDH) expression through directly binding to its 3'UTR. In addition, compared to ECs isolated from normal liver tissues of HCC patients, ECs isolated from tumor tissues expressed markedly low levels of miR-302c but high levels of MTDH. These results suggest that EC-specific miR-302c suppresses tumor growth in HCC through MTDH-mediated inhibition of EndMT. MTDH and miR-302c might provide a new strategy for anti-angiogenic therapy in HCC.

A potential anti-tumor herbal medicine, Corilagin, inhibits ovarian cancer cell growth through blocking the TGF-ß signaling pathways.

BACKGROUND: Phyllanthus niruri L. is a well-known hepatoprotective and antiviral medicinal herb. Recently, we identified Corilagin as a major active component with anti-tumor activity in this herbal medicine. Corilagin is a member of the tannin family that has been discovered in many medicinal plants and has been used as an anti-inflammatory agent. However, there have been few reports of the anti-tumor effects of Corilagin, and its anti-tumor mechanism has not been investigated clearly. The aim of the present study is to investigate the anticancer properties of Corilagin in ovarian cancer cells. METHODS: The ovarian cancer cell lines SKOv3ip, Hey and HO-8910PM were treated with Corilagin and analyzed by Sulforhodamine B (SRB) cell proliferation assay, flow cytometry, and reverse phase protein array (RPPA). Corilagin was delivered intraperitoneally to mice bearing SKOv3ip xenografts. RESULTS: Corilagin inhibited the growth of the ovarian cancer cell lines SKOv3ip and Hey, with IC50 values of less than 30 µM, while displaying low toxicity against normal ovarian surface epithelium cells, with IC50 values of approximately 160 µM. Corilagin induced cell cycle arrest at the G2/M stage and enhanced apoptosis in ovarian cancer cells. Immunoblotting assays demonstrated that Cyclin B1, Myt1, Phospho-cdc2 and Phospho-Weel were down-regulated after Corilagin treatment. Xenograft tumor growth was significantly lower in the Corilagin-treated group compared with the untreated control group (P <0.05). More interestingly, Corilagin inhibited TGF-ß secretion into the culture supernatant of all tested ovarian cancer cell lines and blocked the TGF-ß-induced stabilization of Snail. In contrast, a reduction of TGF-ß secretion was not observed in cancer cells treated with the cytotoxic drug Paclitaxel, suggesting that Corilagin specifically targets TGF-ß secretion. Corilagin blocked the activation of both the canonical Smad and non-canonical ERK/AKT pathways. CONCLUSIONS: Corilagin extracted from Phyllanthus niruri L. acts as a natural, effective therapeutic agent against the growth of ovarian cancer cells via targeted action against the TGF-ß/AKT/ERK/Smad signaling pathways.

Knockdown of mediator complex subunit 19 inhibits the growth of ovarian cancer.

Ovarian cancer causes more deaths than any other type of female reproductive cancer. The development of new therapeutic approaches is required due to the low survival rate using routine methods. The goal of this study was to investigate the effect of the gene silencing of mediator complex subunit 19 (MED19) on cell viability and tumor growth in ovarian cancer. Immunohistochemistry was used to characterize the expression of MED19 in human ovarian cancer tissues. Lentivirus-mediated RNAi was employed to downregulate endogenous MED19 expression in SKOV-3 and HEY ovarian cancer cells. MTT assay, BrdU incorporation assay, colony formation assay, cell cycle analysis and tumor xenografts in nude mice were performed to determine the effects of MED19 silencing on cell viability and tumor growth in vitro and in vivo. The data showed that the expression of MED19 in human ovarian cancer tissues correlated with the level of tumor malignancy. The downregulation of MED19 in ovarian cancer cells significantly inhibited cell proliferation and colony formation in vitro and led to cell cycle arrest in the G0/G1 phase. MED19 RNAi significantly inhibited ovarian cancer tumor growth in engrafted nude mice. Our findings reveal that the knockdown of MED19 by lentivirus-mediated RNAi may be useful in the treatment of human ovarian cancer.

Knockdown of RAB25 promotes autophagy and inhibits cell growth in ovarian cancer cells.

RAB25 belongs to the Rab family of small GTPases and is implicated in the development of various types of human cancer. To evaluate the role of RAB25 in ovarian cancer, RAB25 was knocked down by siRNA in HEY and ES­2 human ovarian cancer cells. Autophagy, cell growth and cell apoptosis were evaluated. The results showed that knockdown of RAB25 increased acidic vesicle organelles and GFP-microtubule-associated protein 1 light chain 3 punctate fluorescence in ovarian cancer cells. Autophagy that promoted by knockdown of RAB25 was not observed in cells where the ERK1/2 signaling pathway had been inhibited by U0126. Knockdown of RAB25 reduced cell cycle progression and cell growth. Apoptosis of ovarian cancer cells could be induced by knockdown of RAB25. These results support the tumorigenic role of RAB25 in ovarian cancer cells.

Re-expression of ARHI (DIRAS3) induces autophagy in breast cancer cells and enhances the inhibitory effect of paclitaxel.

BACKGROUND: ARHI is a Ras-related imprinted gene that inhibits cancer cell growth and motility. ARHI is downregulated in the majority of breast cancers, and loss of its expression is associated with its progression from ductal carcinoma in situ (DCIS) to invasive disease. In ovarian cancer, re-expression of ARHI induces autophagy and leads to autophagic death in cell culture; however, ARHI re-expression enables ovarian cancer cells to remain dormant when they are grown in mice as xenografts. The purpose of this study is to examine whether ARHI induces autophagy in breast cancer cells and to evaluate the effects of ARHI gene re-expression in combination with paclitaxel. METHODS: Re-expression of ARHI was achieved by transfection, by treatment with trichostatin A (TSA) or by a combination of TSA and 5-aza-2'-deoxycytidine (DAC) in breast cancer cell cultures and by liposomal delivery of ARHI in breast tumor xenografts. RESULTS: ARHI re-expression induces autophagy in breast cancer cells, and ARHI is essential for the induction of autophagy. When ARHI was re-expressed in breast cancer cells treated with paclitaxel, the growth inhibitory effect of paclitaxel was enhanced in both the cell culture and the xenografts. Although paclitaxel alone did not induce autophagy in breast cancer cells, it enhanced ARHI-induced autophagy. Conversely, ARHI re-expression promoted paclitaxel-induced apoptosis and G2/M cell cycle arrest. CONCLUSIONS: ARHI re-expression induces autophagic cell death in breast cancer cells and enhances the inhibitory effects of paclitaxel by promoting autophagy, apoptosis, and G2/M cell cycle arrest.

Snail is critical for tumor growth and metastasis of ovarian carcinoma.

Snail, a key inducer of epithelial-mesenchymal transition (EMT), plays an important role in cancer metastasis. To better understand the role of Snail in the metastasis of ovarian carcinoma, expression of Snail was knocked down by antisense-Snail in the highly metastatic ovarian cancer cell line HO8910PM. Gene array analysis revealed that blocking Snail expression suppressed the activity of matrix metalloproteinases (MMPs) and upregulated TIMP3, an MMP inhibitor. These findings suggest that Snail interacts with MMP during tumor invasion and metastasis. In addition, we examined the role of Snail in an ovarian cancer orthotopic model by using the antisense-Snail HO8910PM cell line. We found that the size of primary ovarian cancer tumor and the number of metastatic lesions were significantly reduced when Snail was knocked down. Confirming our initial findings, the activity of MMP2 was greatly inhibited in tumors from antisense-Snail cells. Furthermore, immunohistochemical analysis on ovarian cancer progression tissue array demonstrated that the expression of Snail was significantly higher in metastatic lesions, and Snail expression correlated with the stage of ovarian cancer. Interestingly, in early-stage tumors, Snail was localized in both the cytoplasm and nucleus. In late stage and metastatic lesions, the level of Snail was elevated, and Snail was localized to the nucleus. The expression level and nuclear localization of Snail were also inversely correlated with E-cadherin expression. Overall, our study indicates that Snail plays a critical role in tumor growth and metastasis of ovarian carcinoma through regulation of MMP activity.