冬凌草甲素抗肿瘤作用的研究进展

冬凌草甲素是从唇形科香茶菜属植物中分离出的对映贝壳杉烯二萜类有机化合物,具有广泛的抗肿瘤活性。该文将就冬凌草甲素抗肿瘤活性的化学构效关系及其抗肿瘤机制做一综述。     

Aromatase activity in human ovarian cancer

Eighty-four tumor samples from 70 women with primary ovarian cancer were assayed for cytosol estrogen (ERc) and progestin (PRc) receptor concentrations and aromatase activity. In addition, 22 of the tumors were studied for their response to the aromatase inhibitor, 4-OH-androstenedione, in a soft agar clonogenic cell assay system. Although aromatase activity was detected in almost all of the primary tumors, this enzyme was barely detectable in the majority of metastatic tumor samples. There was no significant correlation between aromatase activity and either the ERc or PRc content of the tumors, or tumor grade. Of 12 tumors grown successfully in the soft agar culture system, only 1 showed a substantial (>50%) reduction in colony-forming efficiency after exposure to the aromatase inhibitor. These results suggest that local estrogen biosynthesis probably does not play an important role in the majority of epithelial ovarian tumors. However, there may be a small subset of estrogen receptor-positive tumors in which aromatase could provide a local growth stimulus.     

Vinculin presents unfavorable prediction in ovarian cancer and prevents proliferation and migration of ovarian cancer cells

The influences of Vinculin on many cancers were blurry, including ovarian cancer. Thus, we concentrated on the efficient role of Vinculin in ovarian cancer and explored the potential mechanism(s). Expression of Vinculin in ovarian cancer tissues and cell lines was investigated by real‐time polymerase chain reaction, immunohistochemistry, and Western blot. The Kaplan–Meier manner with the logrank was performed to assess overall survival. We further evaluated the relations between Vinculin expression and clinicopathological features of ovarian cancer. Moreover, Vinculin was overexpressed or silenced by respectively transfection with pcDNA‐Vinculin or small interfering (si‐Vinculin) into human ovarian cancer cell line Caov3 or human ovarian epithelial cell line (HOEpiC). Thereafter, cell viability, cell apoptosis, and migration were checked by 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2‐H‐tetrazolium bromide, flow cytometer, and scratch assay, respectively. Likewise, the apoptosis‐ and migration‐related proteins were distinguished by Western blot. Compared to the nontumor tissues or HOEpiC cells, Vinculin was significantly lower expressed in the ovarian cancer tissues and cells. Furthermore, we found out that Vinculin was primarily distributed at the cell membrane and cytoplasm. Moreover, Vinculin was negatively associated with International Federation of Gynecology and Obstetrics stage, grade, and distant metastasis. Overexpression of Vinculin dramatically weakened cell viability and migration and stimulated apoptosis. Conversely, suppression of Vinculin showed opposite results. Vinculin presents unfavorable prediction in ovarian cancer and inhibits ovarian cancer proliferation and migration.     

Calcium-mediated telomerase activity in ovarian epithelial cells

Though the potential of telomerase as an anti-cancer target is evident, information about regulation of telomerase remains fragmentary. In the present study, we examined the role of calcium, an essential cellular signaling molecule, in the regulation of telomerase. We found that calcium induced de novo telomerase activity in telomerase-negative ovarian surface epithelial (OSE) cell lines but not in primary cultures of OSE. In addition, we showed that calcium elevated endogenous telomerase levels in a telomerase-positive ovarian cancer cell line. The use of calcium channel blockers or calcium chelators inhibited this calcium-mediated induction of telomerase activity. Furthermore, cadmium and chromium appeared to cause a moderate induction of telomerase activity while several other metal salts did not. Our data provide the first example of calcium-induced telomerase activity in human cell lines, provide a novel avenue for possible intervention of telomerase, and permit development of therapeutic agents for adjunctive chemotherapy.     

Expression and activity of proteases in metastasis of ovarian cancer

The total chymotrypsin-like activity of proteasomes, the activity of 20S- and 26S-proteasome pools and calpains, and the expression of metalloproteinase PAPP-A in primary tumors and metastasized tissues were studied in 13 patients with epithelial ovarian cancer. It was shown that initiation of the process of tumor dissemination occurs against the background of active proteolytic processes. A decrease in activity of 26S-proteasomes and total calpain activity and increased expression of metalloproteinase PAPP-A in the primary tumors were found in patients with ascites as compared with patients without ascites. The disease progression after treatment and achieved stabilization were found in patients with decreased activity of intracellular proteases and a high content of PAPP-A in the primary tumors.     

Presence and role of stem cells in ovarian cancer

Ovarian cancer is the deadliest gynecological malignancy. It is typically diagnosed at advanced stages of the disease, with metastatic sites disseminated widely within the abdominal cavity. Ovarian cancer treatment is challenging due to high disease recurrence and further complicated pursuant to acquired chemoresistance. Cancer stem cell(CSC) theory proposes that both tumor development and progression are driven by undifferentiated stem cells capable of self-renewal and tumor-initiation. The most recent evidence revealed that CSCs in terms of ovarian cancer are not only responsible for primary tumor growth, metastasis and relapse of disease, but also for the development of chemoresistance. As the elimination of this cell population is critical for increasing treatment success, a deeper understanding of ovarian CSCs pathobiology, including epithelial-mesenchymal transition, signaling pathways and tumor microenvironment, is needed. Finally, before introducing new therapeutic agents for ovarian cancer, targeting CSCs, accurate identification of different ovarian stem cell subpopulations, including the very small embryoniclike stem cells suggested as progenitors, is necessary. To these ends, reliable markers of ovarian CSCs should be identified. In this review, we present the current knowledge and a critical discussion concerning ovarian CSCs and their clinical role.     

Primary culture of ovarian surface epithelial cells and ascites-derived ovarian cancer cells from patients

Our laboratory has refined the technique for isolating primary cultures of normal human ovarian surface epithelial (OSE) cells by combining two different protocols involving the enzymatic and mechanical removal of OSE cells from ovarian biopsies. A simple protocol of obtaining primary epithelial ovarian cancer (EOC) cells from the ascites fluid removed from patients with high-grade ovarian cancer is also described. These methods allow for the direct application of many molecular and cellular analyses of normal versus cancer cells isolated freshly from patients, with the added potential for retrospective analyses of archived cells and tissues. Thus, we have included optional steps for the immediate preparation of ascites-derived EOC cells to be used for subsequent cytological analyses. Initial isolation of OSE or EOC cells can be completed in 1 h, and primary cells are further expanded in culture for several weeks.     

Increased DNA methyltransferase activity and DNA methylation following epidermal growth factor stimulation in ovarian cancer cells

Ovarian cancer progression is correlated with accumulation of aberrant CpG island methylation. In ovarian cancer, ascites fluid contains numerous Epidermal-Growth-Factor-Receptor (EGFR) activators, which could result in a tumor microenvironment of constant EGFR activation. Signaling pathways downstream of EGFR, such as Ras, regulate DNA methylation. We hypothesized that chronic EGFR activation could alter DNA methylation. We found that EGFR activation increased DNA methyltransferase (DNMT) activity acutely, as well as after long-term EGF treatment or expression of a mutationally activated EGFR. Furthermore, this increase in DNMT activity was dependent on EGFR catalytic activity and resulted in increased global DNA methylation. Additionally, treatment with the DNMT inhibitor/hypomethylating agent 5-Aza-2’-deoxycytidine (AZA) inhibited the EGF induced increase of both DNMT activity and global methylation. These data support a role for EGFR in the process of accumulated DNA methylation during ovarian cancer progression and suggest that epigenetic therapy may be beneficial for the treatment of ovarian cancer.     

Increased DNA methyltransferase activity and DNA methylation following epidermal growth factor stimulation in ovarian cancer cells

Ovarian cancer progression is correlated with accumulation of aberrant CpG island methylation. In ovarian cancer, ascites fluid contains numerous Epidermal-Growth-Factor-Receptor (EGFR) activators, which could result in a tumor microenvironment of constant EGFR activation. Signaling pathways downstream of EGFR, such as Ras, regulate DNA methylation. We hypothesized that chronic EGFR activation could alter DNA methylation. We found that EGFR activation increased DNA methyltransferase (DNMT) activity acutely, as well as after long-term EGF treatment or expression of a mutationally activated EGFR. Furthermore, this increase in DNMT activity was dependent on EGFR catalytic activity and resulted in increased global DNA methylation. Additionally, treatment with the DNMT inhibitor/hypomethylating agent 5-Aza-2′-deoxycytidine (AZA) inhibited the EGF induced increase of both DNMT activity and global methylation. These data support a role for EGFR in the process of accumulated DNA methylation during ovarian cancer progression and suggest that epigenetic therapy may be beneficial for the treatment of ovarian cancer.Key words: ovarian cancer, DNA methylation, epidermal growth factor receptor, DNA methyltransferase, epigenetics, E-cadherin     

Design,synthesis and antimycobacterial activity evaluation of natural oridonin derivatives

In an effort to develop novel potent antitubercular drugs, thirty-one oridonin derivatives were designed and prepared. All the compounds obtained were screened for their in vitro activities against Mycobacterium phlei, Mycobacterium smegmatis and Mycobacterium marinum. Among them, thirteen compounds showed significant inhibitory activity against M. phlei with MICs less than 2 μg/mL. Compounds 2k, 8d, 10c, 10d containing trans-cinnamic acid moiety were the most potent (MIC = 0.5 μg/mL), comparable to the well-known antitubercular drug streptomycin. The preliminary structure–activity relationships (SARs) were also analyzed.     

BRCA1 regulates follistatin function in ovarian cancer and human ovarian surface epithelial cells

Follistatin (FST), a folliculogenesis regulating protein, is found in relatively high concentrations in female ovarian tissues. FST acts as an antagonist to Activin, which is often elevated in human ovarian carcinoma, and thus may serve as a potential target for therapeutic intervention against ovarian cancer. The breast cancer susceptibility gene 1 (BRCA1) is a known tumor suppressor gene in human breast cancer; however its role in ovarian cancer is not well understood. We performed microarray analysis on human ovarian carcinoma cell line SKOV3 that stably overexpress wild-type BRCA1 and compared with the corresponding empty vector-transfected clones. We found that stable expression of BRCA1 not only stimulates FST secretion but also simultaneously inhibits Activin expression. To determine the physiological importance of this phenomenon, we further investigated the effect of cellular BRCA1 on the FST secretion in immortalized ovarian surface epithelial (IOSE) cells derived from either normal human ovaries or ovaries of an ovarian cancer patient carrying a mutation in BRCA1 gene. Knock-down of BRCA1 in normal IOSE cells demonstrates down-regulation of FST secretion along with the simultaneous up-regulation of Activin expression. Furthermore, knock-down of FST in IOSE cell lines as well as SKOV3 cell line showed significantly reduced cell proliferation and decreased cell migration when compared with the respective controls. Thus, these findings suggest a novel function for BRCA1 as a regulator of FST expression and function in human ovarian cells.     

Clinical significance of side population in ovarian cancer cells

Recently, accumulating evidence has suggested that tumors, including ovarian cancer, are composed of a heterogeneous cell population with a small subset of cancer stem cells (CSCs) that sustain tumor formation and growth. The emergence of drug resistance is one of the most difficult problems in the treatment of ovarian cancer, which has been explained recently by the potential of CSCs to have superior resistance against anti-cancer drugs than conventional cancer cells. In this study, we expanded this line of study to examine whether this phenomenon is also observed in clinical specimens of ovarian cancer cells. In total we could analyze 28 samples out of 60 obtained from ovarian cancer patients. The clinical samples were subjected to testing of the expression of side population (SP) as a CSC marker, and according to the presence of SP (SP+) or absence of SP (SP-), clinicopathological significances were analyzed. Although there was no statistical significance, there were more SP+s in recurrent cases as well as in ascitic and peritoneal dissemination than in primary tumor of the ovary. There was no correlation between SP status and FIGO staging. In 19 cases of those who could be followed more than 6 months from initial therapy, there were 8 cases of recurrence or death from disease, and all of these were SP+. On the other hand, in 11 cases of disease-free survivors, 6 were SP+. There was a significant difference in prognosis between SP+ and SP- (p = 0.017). Although this study was limited, it revealed that SP could be contained more in recurrent or metastatic tumors than in primary tumors, and also that the presence of SP could be a risk factor of recurrence in ovarian cancer. Therefore, a novel therapeutic strategy targeting SP could improve the prognosis of ovarian cancer.     

Role of ether-linked lysophosphatidic acids in ovarian cancer cells

Naturally occurring alkyl- and alkenyl-lysophosphatidic acids (al-LPAs) are detected and elevated in ovarian cancer ascites compared with ascites from non-malignant diseases. Here we describe the biological functions and signaling properties of these ether-linked LPAs in ovarian cancer cells. They are elevated and stable in ovarian cancer ascites, which represents an in vivo environment for ovarian cancer cells. They stimulated DNA synthesis and proliferation of ovarian cancer cells. In addition, they induced cell migration and the secretion of a pro-angiogenic factor, interleukin-8 (IL-8), in ovarian cancer cells. The latter two processes are potentially related to tumor metastasis and angiogenesis, respectively. Al-LPAs induced diverse signaling pathways in ovarian cancer cells. Their mitogenic activity depended on the activation of the G(i/o) protein, phosphatidylinositol-3 kinase (PI3K), and mitogen-activated protein (MAP) kinase kinase (MEK), but not p38 mitogen activated protein kinase (MAP kinase). S473 phosphorylation of protein kinase B (Akt) by these lipids required activation of the G(i/o) protein, PI3K, MEK, p38 MAP kinase, and Rho. However, T308 phosphorylation of Akt stimulated by al-LPAs did not require activation of p38 MAP kinase. On the other hand, cell migration induced by al-LPAs depended on activities of the G(i/o) protein, PI3K, and Rho, but not MEK. These data suggest that ether-linked LPAs may play an important role in ovarian cancer development.     

Secretion of annexin A3 from ovarian cancer cells and its association with platinum resistance in ovarian cancer patients

Early detection of resistance to platinum-based therapy is critical for improving the treatment of ovarian cancers. We have previously found that increased expression of annexin A3 is a mechanism for platinum resistance in ovarian cancer cells. Here we demonstrate that annexin A3 can be detected in the culture medium of ovarian cancer cells, particularly these cells that express high levels of annexin A3. Levels of annexin A3 were then determined in sera from ovarian cancer patients using an enzyme-linked immunosorbent assay. Compared with those from normal donors, sera from ovarian cancer patients contain significantly higher levels of annexin A3. Furthermore, serum levels of annexin A3 were significantly higher in platinum-resistant patients than in platinum-sensitive patients. To gain insight into the mechanism of secretion, the ovarian cancer cell lines were examined using both transmission electron microscopy and immunoelectron microscopy. Compared with parent cells, there are significantly more vesicles in the cytoplasm of ovarian cancer cells that express high levels of annexin A3, and at least some vesicles are annexin A3-positive. Moreover, some vesicles appear to be fused with the cell membrane, suggesting that annexin A3 secretion may be associated with exocytosis and the release of exosomes. This is supported by our observation that ovarian cancer cells expressing higher levels of annexin A3 released increased numbers of exosomes. Furthermore, annexin A3 can be detected in exosomes released from cisplatin-resistant cells (SKOV3/Cis) by immunoblotting and immunoelectron microscopy.     

Orotic acid induces apoptotic death in ovarian adult granulosa tumour cells and increases mitochondrial activity in normal ovarian granulosa cells

Orotic acid (OA) is a natural product that acts as a precursor in the pyrimidine nucleotide biosynthesis pathway. Most studies concerning administration of OA focus on its therapeutic effects; however, its effect on tumours is unclear. We aimed to determine whether treatment with OA influences the viability and apoptosis of normal (HGrC1) and tumour-derived (KGN) human ovarian granulosa cells. The effects of OA (10–250 μM) on viability and apoptosis of both cell lines were determined by using alamarBlue and assessing caspase-3/7 activity, respectively. Annexin V binding and loss of membrane integrity were evaluated in KGN cells. The cell cycle and proliferation of HGrC1 cells were assessed by performing flow cytometric and DNA content analyses, respectively. The influence of OA (10 and 100 μM) on cell cycle- and apoptosis-related gene expression was assessed by RT-qPCR in both cell lines. Mitochondrial activity was analysed by JC-1 staining in HGrC1 cells. In KGN cells, OA reduced viability and increased caspase-3/7 activity, but did not affect mRNA expression of Caspase 3, BAX, and BCL2. OA enhanced proliferation and mitochondrial activity in HGrC1 cells without activating apoptosis. This study demonstrates that the anti-cancer properties of OA in ovarian granulosa tumour cells are not related to changes in apoptosis-associated gene expression, but to increased caspase-3/7 activity. Thus, OA is a promising therapeutic agent for ovarian granulosa tumours. Further, our results suggest that differences in basal expression of cell cycle- and apoptosis-related genes between the two cell lines are responsible for their different responses to OA.     

Estrogen regulated proteases and antiproteases in ovarian and breast cancer cells

Cathepsin D (cath-D), an estrogen-regulated protease appears mostly to increase the number of tumor cells rather than their invasion or motility through the extracellular matrix. Estradiol is mitogenic but in vitro it also inhibits invasion and motility. In this review, we discuss the mechanism of this inhibition and the hormonal regulation of other proteases and protease inhibitors possibly involved in the control of tumor cell invasion by estrogens.