Ganoderma lucidum inhibits proliferation and induces apoptosis in human prostate cancer cells PC-3

Ganoderma lucidum (Reishi), an oriental medical mushroom, has been widely used in Asian countries for centuries to prevent or treat different diseases, including cancer. However, the mechanism(s) responsible for the effects of Ganoderma lucidum on cancer cells remain to be elucidated. We have previously demonstrated that Ganoderma lucidum down-regulated the expression of NF-kappaB-regulated urokinase plasminogen activator (uPA) and uPA receptor (uPAR), which resulted in suppression of cell migration of highly invasive human breast and prostate cancer cells. In this study, we investigated the effects of Ganoderma lucidum on cell proliferation, cell cycle, and apoptosis in human prostate cancer cells PC-3. Our data demonstrate that Ganoderma lucidum inhibits cell proliferation in a dose- and time-dependent manner by the down-regulation of expression of cyclin B and Cdc2 and by the up-regulation of p21 expression. The inhibition of cell growth was also demonstrated by cell cycle arrest at G2/M phase. Furthermore, Ganoderma lucidum induced apoptosis of PC-3 cells with a slight decrease in the expression of NF-kappaB-regulated Bcl-2 and Bcl-xl. However, the expression of proapoptotic Bax protein was markedly up-regulated, resulting in the enhancement of the ratio of Bax/Bcl-2 and Bax/Bcl-xl. Thus, Ganoderma lucidum exerts its effect on cancer cells by multiple mechanisms and may have potential therapeutic use for the prevention and treatment of cancer.     

Differential cellular responses induced by dorsomorphin and LDN‐193189 in chemotherapy‐sensitive and chemotherapy‐resistant human epithelial ovarian cancer cells

Inherent or acquired drug resistance is a major contributor to epithelial ovarian cancer (EOC) mortality. Novel drugs or drug combinations that produce EOC cell death or resensitize drug resistant cells to standard chemotherapy may improve patient treatment. After conducting drug tolerability studies for the multikinase inhibitors dorsomorphin (DM) and it is structural analogue LDN‐193189 (LDN), these drugs were tested in a mouse intraperitoneal xenograft model of EOC. DM significantly increased survival, whereas LDN showed a trend toward increased survival. In vitro experiments using cisplatin (CP)‐resistant EOC cell lines, A2780‐cp or SKOV3, we determined that pretreatment or cotreatment with DM or LDN resensitized cells to the killing effect of CP or carboplatin (CB). DM was capable of blocking EOC cell cycle and migration, whereas LDN produced a less pronounced effect on cell cycle and no effect on migration. Subsequent analyses using primary human EOC cell samples or additional established EOC cells lines showed that DM or LDN induced a dose‐dependent autophagic or cell death response, respectively. DM induced a characteristic morphological change with the appearance of numerous LC3B‐containing acidic vacuoles and an increase in LC3BII levels. This was coincident with a decrease in cell growth and the altered cell cycle consistent with DM‐induced cytostasis. By contrast, LDN produced a caspase 3‐independent, reactive oxygen species‐dependent cell death. Overall, DM and LDN possess drug characteristics suitable for adjuvant agents used to treat chemotherapy‐sensitive and ‐resistant EOC.     

Combination of nigericin with cisplatin enhances the inhibitory effect of cisplatin on epithelial ovarian cancer metastasis by inhibiting slug expression via the Wnt/β-catenin signalling pathway

Epithelial ovarian cancer (EOC) is the most lethal cancer among female genital tumours. Standard therapies, including postoperative chemotherapy, exhibit high proportions of recurrence and resistance. Novel therapeutic strategies are combined with chemotherapy. Emerging studies have demonstrated that nigericin, an H⁺, K⁺ and Pb²⁺ ionophore, exhibits promising anticancer activity in various types of malignancy, such as colorectal and epithelial ovarian cancer. Our previous study suggested that nigericin could regulate EOC cell proliferation, migration and invasion, and may be a novel chemotherapy candidate for EOC. However, to the best of our knowledge, the effects of combined therapy with cisplatin, and the associated underlying mechanisms, are not yet fully understood. The present study aimed to clarify the effects of combined chemical therapy with nigericin and cisplatin on EOC cells and to reveal its mechanism. Wound healing, Transwell, cell viability and colony formation assays were used to measure the migration, invasion and proliferation of EOC cells. Western blotting was used to detect protein expression. A slug overexpression lentivirus was used to create a slug overexpression model in SK-OV-3 cells. Small interfering RNA was used to knock down slug expression. Nigericin combined with cisplatin enhanced the inhibitory effects of cisplatin on the migration and colony formation of EOC cells. Nigericin also enhanced the inhibitory effects of cisplatin on the expression levels of MMP7, as well as the inhibitory effects of cisplatin on the expression levels of β-catenin and GSK-3β, indicating that nigericin and cisplatin regulated in the Wnt/β-catenin signalling pathway. When slug was knocked down, the effect of nigericin was weakened. Overexpression of slug could repress the inhibitory effect of nigericin on the Wnt/β-catenin signalling pathway. Furthermore, nigericin inhibited slug expression by enhancing its modification through small ubiquitin-like modifiers (SUMOs; referred to as SUMOylation). Overall, the present results demonstrated that nigericin combined with cisplatin might serve as a novel therapeutic strategy in patients with metastatic EOC because the combined therapy had higher effectiveness than single drug use. The underlying mechanism of combined therapy maybe the enhanced inhibitory effect of slug through its nigericin-induced SUMOylation.     

Combined effect of green tea and Ganoderma lucidum on invasive behavior of breast cancer cells

Epidemiological studies have suggested that consumption of green tea may decrease the risk of a variety of cancers. In addition, mushroom Ganoderma lucidum has been used for the promotion of health, longevity and treatment of cancer in traditional Chinese medicine. In the present study we show that extract from green tea (GTE) increased the anticancer effect of G. lucidum extract (GLE) on cell proliferation (anchorage-dependent growth) as well as colony formation (anchorage-independent growth) of breast cancer cells. This effect was mediated by the down-regulation of expression of oncogene c-myc in MDA-MB-231 cells. Although individual GTE and GLE independently inhibited adhesion, migration and invasion of MDA-MB-231 cells, their combination demonstrated a synergistic effect, which was mediated by the suppression of secretion of urokinase plasminogen activator (uPA) from breast cancer cells. Our study suggests the potential use of combined green tea and G. lucidum extracts for the suppression of growth and invasiveness of metastatic breast cancers.     

miR-483-5p在上皮性卵巢癌中的表达及其对顺铂敏感性的影响

背景与目的：顺铂是目前临床上治疗上皮性卵巢癌的一线化疗药物之一,但许多患者对铂类药物耐药。miR-483-5p在肺癌中过表达,然而目前尚未见miR-483-5p在上皮性卵巢癌中的研究。该研究检测miR-483-5p在上皮性卵巢癌组织和上皮性卵巢癌细胞系中的表达并探讨其对上皮性卵巢癌细胞对顺铂敏感性的影响。方法：采用实时荧光定量聚合酶链反应(real-time fluorescent quantitative polymerase chain reaction,RTFQ-PCR)检测43例上皮性卵巢癌患者的肿瘤组织、8例正常卵巢组织和5种上皮性卵巢癌细胞系中miR-483-5p的表达情况;通过慢病毒上调或敲低卵巢癌细胞miR-483-5p表达,应用CCK-8实验检测miR-483-5p对上皮性卵巢癌细胞系顺铂敏感性的影响。结果：上皮性卵巢癌组织中miR-483-5p表达明显高于正常卵巢组织(P<0.01)。此外,miR-483-5p在晚期上皮性卵巢癌组织中的表达水平显著高于早期肿瘤组织(P<0.05)。5种上皮性卵巢癌细胞系中SKOV3细胞表达miR-483-5p的量最低;miR-483-5p在上皮性卵巢癌顺铂耐药A2780/CP细胞中表达量最高。上调SKOV3细胞中miR-483-5p的表达能够降低上皮性卵巢癌细胞对顺铂的敏感性,并下调p21及Bcl-2的表达;下调A2780/CP细胞miR-483-5p的表达能够增加细胞对顺铂的敏感性,并上调p21及Bcl-2的表达。结论：miR-483-5p在上皮性卵巢癌组织中高表达并对顺铂耐药,可以作为临床预测上皮性卵巢癌对顺铂敏感性的生物标志物之一。     

Sensitization of ovarian cancer cells to cisplatin by gold nanoparticles

Recently we reported that gold nanoparticles (AuNPs) inhibit ovarian tumor growth and metastasis in mice by reversing epithelial-mesenchymal transition (EMT). Since EMT is known to confer drug resistance to cancer cells, we wanted to investigate whether anti-EMT property of AuNP could be utilized to sensitize ovarian cancer cells to cisplatin. Herein, we report that AuNPs prevent cisplatin-induced acquired chemoresistance and stemness in ovarian cancer cells and sensitize them to cisplatin. AuNPs inhibit cisplatin induced EMT, decrease the side population cells and key stem cell markers such as ALDH1, CD44, CD133, Sox2, MDR1 and ABCG2 in ovarian cancer cells. Mechanistically, AuNPs prevent cisplatin-induced activation of Akt and NF-κB signaling axis in ovarian cancer cells that are critical for EMT, stem cell maintenance and drug resistance. In vivo, AuNPs sensitize orthotopically implanted ovarian tumor to a low dose of cisplatin and significantly inhibit tumor growth via facilitated delivery of both AuNP and cisplatin. These findings suggest that by depleting stem cell pools and inhibiting key molecular pathways gold nanoparticles sensitize ovarian cancer cells to cisplatin and may be used in combination to inhibit tumor growth and metastasis in ovarian cancer.     

选择性环氧化酶-2抑制剂对卵巢癌细胞抑制作用的体内体外研究

目的:研究选择性环氧化酶-2抑制剂美洛昔康在体内体外对卵巢癌生长侵袭的抑制作用。方法:使用2种卵巢癌细胞株,研究不同浓度的美洛昔康对于卵巢癌细胞增殖的抑制作用。利用移植卵巢癌细胞株小鼠模型,研究美洛昔康对小鼠背部皮下移植瘤生长以及肿瘤中VEGF表达、微血管密度以及细胞凋亡的影响,并将之与顺铂的作用相比较。结果:在体外实验中,美洛昔康对2种卵巢癌细胞的增殖均有抑制作用,且呈剂量依赖性。体内实验证明美洛昔康明显抑制小鼠皮下移植瘤的生长,其抑制率为30.79%（P＜ 0.0001）。各组肿瘤组织中VEGF的表达经半定量测定分别为:每高倍视野对照组5.17±0.52、、顺铂组4.32±0.73及美洛昔康组4.81±0.58。与对照组相比,美洛昔康饲养的小鼠肿瘤中VEGF的表达受到明显抑制（P＜0.0001）并伴随微血管密度的明显减低。TUNEL法对肿瘤组织中凋亡细胞计数,结果分别为:对照组12.8±1.3、顺铂组11.5±1.8及美洛昔康组23.3±2.7。与对照组比较,美洛昔康饲养的小鼠肿瘤中,细胞凋亡的数量明显增多（P＜0.001）。美洛昔康的抗肿瘤效果在某些方面优于顺铂。结论:美洛昔康在体内体外实验中表现出抗卵巢肿瘤生长和侵袭的作用。选择性环氧化酶-2抑制剂或许可以作为潜在的新型抗卵巢癌药物应用于临床。     

Midkine as a potential diagnostic marker in epithelial ovarian cancer for cisplatin/paclitaxel combination clinical therapy

The paclitaxel/cisplatin combination therapy commonly is used as the first-line treatment for advanced ovarian cancer patients. Midkine (MK), known as a novel tumor biomarker, has been elevated in the serum of patients with epithelial ovarian cancer (EOC). In this study, we aimed to detect the expression of MK in EOC tissues and evaluate clinical value of MK in diagnosis and therapy of EOC. We perform immunohistochemistry analysis to detect MK in EOC sample with postoperative platinum/paclitaxel combination therapy, we found that 71.4% (85 in 119 samples) of these samples were MK positive (> 10% of the cells were stained), and the expression of MK was significantly associated with disease histology (P = 0.038) as well as differentiation grade (P < 0.001). Moreover, MK positive samples show much more sensitive to cisplatin/paclitaxel combination therapy, compared with MK negative samples (P = 0.029). Those results indicated that MK expression might correlate with paclitaxel and/or cisplatin cytotoxicity in clinical therapy of EOC. Then, we evaluated the sensitivity to cisplatin and paclitaxel in 5 ovarian cancer cell lines (ES2, A2870, HO-8910, SKOV3 and SW626), and ES2, the highest MK expression among those cell lines, show the most sensitive to paclitaxel and cisplatin. Further, we confirmed this correlation between MK and paclitaxel and/or cisplatin cytotoxicity with the gain- and lost- of function. Finally, we demonstrated that MK enhanced the cytotoxicity of paclitaxel and/or cisplatin by accumulated cisplatin and paclitaxel through inhibited the expression of multidrug resistance-associated protein 3 (MRP3). In conclusion, MK could be an effective biomarker in diagnosis and therapy of EOC, especially for the drug selection at the time of initial diagnosis.     

Expression and functions of galectin-7 in ovarian cancer

There is a critical need to develop effective new strategies for diagnosis and treatment of ovarian cancer. In the present work, we investigated the expression of galectin-7 (gal-7) in epithelial ovarian cancer (EOC) cells and studied its functional relevance. Immunohistochemical analysis of gal-7 expression in tissue microarrays showed that while gal-7 was not detected in normal ovarian tissues, positive cytoplasmic staining of gal-7 was detected in epithelial cells in all EOC histological subtypes but was more frequent in high grade tumors and metastatic samples. Gal-7 expression correlated with a significant difference in the overall survival of patients with ovarian serous cystadenocarcinoma. Furthermore, using human EOC cell lines, we found that gal-7 expression was induced by mutant p53. Mechanistically, Matrigel invasion assays and live cell imaging showed that gal-7 increased the invasive behavior of ovarian cancer cells by inducing MMP-9 and increasing cell motility. EOC cells can also secrete gal-7. Recombinant human gal-7 kills Jurkat T cells and human peripheral T cells, suggesting that gal-7 also has immunosuppressive properties. Taken together, our study validates the clinical significance of gal-7 overexpression in ovarian cancer and provides a rationale for targeting gal-7 to improve the outcome of patients with this disease.     

Sensitizing ovarian cancer cells to chemotherapy by interfering with pathways that are involved in the formation of cancer stem cells

Chemotherapy often fails to eradicate cancer stem cells (CSCs) that drive cancer recurrence. In fact, the treated tumors often contain a higher frequency of chemo-resistant CSCs. It is thought that CSC formation is supported by exposure of cancer cells to sub-cytotoxic chemotherapy doses as a result of poor drug penetration in epithelial tumors. We have shown that low-dos cisplatin triggers the transdifferentiation of ovarian cancer cells into CSCs through processes that are also involved in the generation and maintenance of induced pluripotent stem (iPS) cells. Considering similarities between CSCs and iPS cells, we screened a library of 60 synthetic small-molecule compounds, designed to influence EMT/MET signaling in iPS cells on primary ovarian cancer cells. Using a Nanog reporter system we identified a series of compounds capable of blocking the cisplatin triggered formation of CSCs. We then focused on compound GHDM-1515, a drug that acts on pathways that regulate histone demethylases. We demonstrated that co-treatment of primary ovarian cancer cells with GHDM-1515 significantly increased cisplatin induced apoptosis, specifically apoptosis of CSCs. GHDM-1515 inhibited EMT and the cisplatin-induced formation of CSCs. This suggests that GHDM-1515 can sensitize ovarian cancer cells to low-dose cisplatin and potentially enhance the efficacy of cisplatin chemotherapy.     

Ganoderma lucidum extracts inhibit growth and induce actin polymerization in bladder cancer cells in vitro

This study was conducted to investigate chemopreventive effects of Ganoderma lucidum using a unique in vitro human urothelial cell (HUC) model consisted of HUC-PC cells and MTC-11 cells. Ethanol and water extracts of fruiting bodies and spores of the G. lucidum were used to examine growth inhibition, actin polymerization status, and impact of actin remodeling on cell migration and adhesion. Results showed that ethanol extracts had a stronger growth inhibition effect than water extracts. Cell cycle analysis showed that the growth inhibition effect was associated with G2/M arrest. At non-cytotoxic concentrations (40-80 microg/ml), these extracts induced actin polymerization, which in turn inhibited carcinogen 4-aminobiphenyl induced migration in both cell lines. The increased actin polymerization was associated with increased stress fibers and focal adhesion complex formation, however, expression of matrix metalloproteinase-2 and focal adhesion kinase (total and phospholated) were unchanged, which suggests that other mechanisms may be involved.     

人上皮性卵巢癌组织中PFTK1的表达及其对细胞迁移、侵袭及增殖能力的影响

目的:研究PFTAIRE蛋白激酶1（PFTAIRE protein kinase 1,PFTK1）在人上皮性卵巢癌（epithelial ovarian cancer,EOC）组织中的表达,以及PFTK1基因的沉默对人卵巢癌细胞迁移、侵袭及增殖能力的影响。方法: 随机选取我院2015年6月至2016年11月收治的25例EOC患者为研究对象,采用Western blot检测癌组织以及癌旁组织中PFTK1蛋白的表达情况。选取PFTK1高表达的人卵巢癌细胞株SKOV3为研究对象,转染PFTK1-siRNA后,采用流式细胞仪检测PFTK1基因的沉默对SKOV3细胞周期的影响,采用细胞迁移和侵袭实验（Transwell）检测SKOV3细胞迁移、侵袭能力的变化,采用平板克隆形成实验检测SKOV3细胞增殖能力的变化。结果:Western blot检测结果显示,EOC患者癌组织中的PFTK1蛋白表达量显著高于癌旁组织（P＜0.01）;流式细胞仪检测结果显示,PFTK1-siRNA组细胞出现明显的生长抑制,细胞多停滞在G0/G1期,与对照组相比差异显著（P＜0.01）;细胞迁移和侵袭实验（Transwell）结果显示,PFTK1-siRNA组细胞的迁移和侵袭能力与对照组比较显著下降,且差异均显著（P＜0.01）;平板克隆形成实验结果显示,PFTK1-siRNA组细胞增殖能力显著降低,与对照组相比差异显著（P＜0.01）。结论:PFTK1在人EOC癌组织中呈现高表达状态;PFTK1基因沉默后,EOC细胞株SKOV3的细胞迁移、侵袭及增殖能力均受到不同程度的抑制,这将为基因治疗人卵巢癌提供一定的理论依据。     

Cyclooxygenase-1 is a potential target for prevention and treatment of ovarian epithelial cancer

The precise genetic and molecular defects underlying epithelial ovarian cancer (EOC) remain largely unknown, and treatment options for patients with advanced disease are limited. Cyclooxygenases (COX-1 and COX-2) catalyze the conversion of arachidonic acid to prostaglandins. Whereas overwhelming evidence suggests a role for COX-2 in a variety of cancers, the contribution of COX-1 remains much less explored. The expression status of COX isoforms in ovarian cancers also remains confusing. We have previously shown that human epithelial ovarian tumors have increased levels of COX-1 but not COX-2. To more carefully examine the role of COXs in ovarian cancer, we used a mouse model of EOC in which genetic and oncogenic modifications were experimentally engineered into ovarian surface epithelial cells (OSE) thought to be the cells of origin for human EOC. These OSE cells produce tumors when allografted into host mice. Using multiple approaches, we observed that OSE cells and the tumors comprised of these cells express high levels of COX-1 but not COX-2. Prostacyclin (PGI(2)) is the major prostaglandin generated downstream of COX-1 in these cells, and SC-560, a COX-1-selective inhibitor, dramatically inhibits PGI(2) production. More importantly, SC-560 reduced the growth of tumors when OSE cells were allografted in nude female mice. In contrast, the COX-2-selective inhibitor celecoxib had little effect on tumor growth. The growth inhibitory effects of SC-560 result from reduced cell proliferation and/or accelerated apoptosis. Our results imply COX-1 as a target for the prevention and/or treatment of EOC.     

Role of the polypeptide N-acetylgalactosaminyltransferase 3 in ovarian cancer progression: possible implications in abnormal mucin O-glycosylation

Previously, we have identified the polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3) gene as notably hypomethylated in low-malignant potential (LMP) and high-grade (HG) serous epithelial ovarian tumors, compared to normal ovarian tissues. Here we show that GALNT3 is strongly overexpressed in HG serous EOC tumors as compared to normal ovarian tissue. Moreover, the GALNT3 expression significantly correlated with shorter progression-free survival (PFS) intervals in epithelial ovarian cancer (EOC) patients with advanced disease.Knockdown of the GALNT3 expression in EOC cells led to sharp decrease of cell proliferation and induced S-phase cell cycle arrest. Additionally, GALNT3 suppression significantly inhibited EOC cell migration and invasion. Gene expression profiling and consecutive network and pathway analyses confirmed these findings, as numerous genes and pathways known previously to be implicated in ovarian tumorigenesis, including EOC tumor invasion and metastasis, were found to be downregulated upon GALNT3 suppression, while some tumor suppressor genes were induced. Moreover, GALNT3 downregulation was associated with reduced MUC1 protein expression in EOC cells, probably related to destabilization of the MUC1 protein due to lack of GALNT3 glycosylation activity. GALNT3 knockdown was also accompanied with increase of the cell adhesion molecules β-catenin and E-cadherin, which are normally suppressed by MUC1 in cancer, thus supporting the role of the GALNT3-MUC1 axis in EOC invasion.Taken together, our data are indicative for a strong oncogenic potential of the GALNT3 gene in advanced EOC and identify this transferase as a novel EOC biomarker and putative EOC therapeutic target. Our findings also suggest that GALNT3 overexpression might contribute to EOC progression through aberrant mucin O-glycosylation     

PDGF BB induces VEGF secretion in ovarian cancer

We identified the platelet derived growth factor receptor (PDGFR) as a potential target in epithelial ovarian carcinoma (EOC). This led us to test whether inhibition of the PDGFR affects ovarian cancer cell proliferation and survival and regulates other processes critical to tumor growth and metastasis. We postulated that there is a correlation between the PDGF-PDGFR axis and the secretion of VEGF in EOC. VEGF secretion in ovarian tumors, cancer cells, serum and ascites fluid was measured by IHC, Western Blot and ELISA. We found increased VEGF expression and secretion in most ovarian tumors (by IHC), in EOC malignant ascites and in the conditioned media of primary ovarian cancer cells (quantified by ELISA). In malignant ascites, the levels of secreted PDGF BB and VEGF were strongly correlated (Pearson coefficient of correlation R = 0.728), suggesting that the two pathways interconnect. In PDGFR expressing immortalized ovarian cancer cells, PDGF potently induced VEGF secretion, while imatinib mesylate (Gleevec), a partially selective PDGFR inhibitor, reduced PDGF stimulated VEGF production to basal state. In ovarian cancer cells overexpressing constitutively active Akt, imatinib inhibited partially VEGF secretion, suggesting that the PI3K/Akt pathway is implicated in PDGF-stimulated VEGF secretion. In summary, these results suggest a correlation between the PDGF and VEGF networks in ovarian cancer cells and tumors. The effects of imatinib on VEGF secretion in tumor cells may affect the tumor microenvironment in a manner detrimental to tumor progression.