MT-4 Suppresses Resistant Ovarian Cancer Growth through Targeting Tubulin and HSP27

Objective

In this study, the anticancer mechanisms of MT-4 were examined in A2780 and multidrug-resistant NCI-ADR/res human ovarian cancer cell lines.

Methods

To evaluate the activity of MT-4, we performed in vitro cell viability and cell cycle assays and in vivo xenograft assays. Immunoblotting analysis was carried out to evaluate the effect of MT-4 on ovarian cancer. Tubulin polymerization was determined using a tubulin binding assay.

Results

MT-4 (2-Methoxy-5-[2-(3,4,5-trimethoxy-phenyl)-ethyl]-phenol), a derivative of moscatilin, can inhibit both sensitive A2780 and multidrug-resistant NCI-ADR/res cell growth and viability. MT-4 inhibited tubulin polymerization to induce G2/M arrest followed by caspase-mediated apoptosis. Further studies indicated that MT-4 is not a substrate of P-glycoprotein (p-gp). MT-4 also caused G2/M cell cycle arrest, accompanied by the upregulation of cyclin B, p-Thr161 Cdc2/p34, polo-like kinase 1 (PLK1), Aurora kinase B, and phospho-Ser10-histone H3 protein levels. In addition, we found that p38 MAPK pathway activation was involved in MT-4-induced apoptosis. Most importantly, MT-4 also decreased heat shock protein 27 expression and reduced its interaction with caspase-3, which inured cancer cells to chemotherapy resistance. Treatment of cells with SB203580 or overexpression of dominant negative (DN)-p38 or wild-type HSP27 reduced PARP cleavage caused by MT-4. MT-4 induced apoptosis through regulation of p38 and HSP27. Our xenograft models also show the in vivo efficacy of MT-4. MT-4 inhibited both A2780 and NCI-ADR/res cell growth in vitro and in vivo.

Conclusion

These findings indicate that MT-4 could be a potential lead compound for the treatment of multidrug-resistant ovarian cancer.     

Withaferin A Alone and in Combination with Cisplatin Suppresses Growth and Metastasis of Ovarian Cancer by Targeting Putative Cancer Stem Cells

Currently, the treatment for ovarian cancer entails cytoreductive surgery followed by chemotherapy, mainly, carboplatin combined with paclitaxel. Although this regimen is initially effective in a high percentage of cases, unfortunately within few months of initial treatment, tumor relapse occurs because of platinum-resistance. This is attributed to chemo-resistance of cancer stem cells (CSCs). Herein we show for the first time that withaferin A (WFA), a bioactive compound isolated from the plant Withania somnifera, when used alone or in combination with cisplatin (CIS) targets putative CSCs. Treatment of nude mice bearing orthotopic ovarian tumors generated by injecting human ovarian epithelial cancer cell line (A2780) with WFA and cisplatin (WFA) alone or in combination resulted in a 70 to 80% reduction in tumor growth and complete inhibition of metastasis to other organs compared to untreated controls. Histochemical and Western blot analysis of the tumors revealed that inclusion of WFA (2 mg/kg) resulted in a highly significant elimination of cells expressing CSC markers - CD44, CD24, CD34, CD117 and Oct4 and downregulation of Notch1, Hes1 and Hey1 genes. In contrast treatment of mice with CIS alone (6 mg/kg) had opposite effect on those cells. Increase in cells expressing CSC markers and Notch1 signaling pathway in tumors exposed to CIS may explain recurrence of cancer in patients treated with carboplatin and paclitaxel. Since, WFA alone or in combination with CIS eliminates putative CSCs, we conclude that WFA in combination with CIS may present more efficacious therapy for ovarian cancer.     

New Blocking Antibodies Impede Adhesion,Migration and Survival of Ovarian Cancer Cells,Highlighting MFGE8 as a Potential Therapeutic Target of Human Ovarian Carcinoma

Milk Fat Globule – EGF – factor VIII (MFGE8), also called lactadherin, is a secreted protein, which binds extracellularly to phosphatidylserine and to αvβ3 and αvβ5 integrins. On human and mouse cells expressing these integrins, such as endothelial cells, phagocytes and some tumors, MFGE8/lactadherin has been shown to promote survival, epithelial to mesenchymal transition and phagocytosis. A protumoral function of MFGE8 has consequently been documented for a few types of human cancers, including melanoma, a subtype of breast cancers, and bladder carcinoma. Inhibiting the functions of MFGE8 could thus represent a new type of therapy for human cancers. Here, we show by immunohistochemistry on a collection of human ovarian cancers that MFGE8 is overexpressed in 45% of these tumors, and we confirm that it is specifically overexpressed in the triple-negative subtype of human breast cancers. We have established new in vitro assays to measure the effect of MFGE8 on survival, adhesion and migration of human ovarian and triple-negative breast cancer cell lines. Using these assays, we could identify new MFGE8-specific monoclonal antibodies, which efficiently blocked these three tumor-promoting effects of MFGE8. Our results suggest future use of MFGE8-blocking antibodies as new anti-cancer therapeutics in subgroups of ovarian carcinoma, and triple-negative breast carcinoma patients.     

The Influences of a Targeting Peptide on the Ovarian Cancer Cell Motility

Tumor metastasis is the major cause of death from ovarian cancer. Therefore, targeted therapy, which could prevent the ability of cells to metastasize to different organs, is an effective treatment for ovarian cancer at present. Previous study indicated that the peptide WSGPGVWGASVK (WSG) inhibited the adhesive ability of SK-OV-3 to extracellular matrix. For further study, we have investigated the effects of the peptide WSG on the ovarian cancer cell migration. Results showed that WSG peptide promoted the migration of SK-OV-3 and HUVEC cells through regulating the expression of talin and (p)-paxilin protein. Besides, WSG peptide inhibited the SK-OV-3 viability. The expression of human matrix metalloproteinase MMP-2 and MMP-9 of SK-OV-3 cells was inhibited. All these results suggested that peptide WSG might be used in ovarian cancer therapy.     

MicroRNA-137 Contributes to Dampened Tumorigenesis in Human Gastric Cancer by Targeting AKT2

MiRNAs play important roles in tumorigenesis. This study focused on exploring the effects and regulation mechanism of miRNA-137 on the biological behaviors of gastric cancer. Total RNA was extracted from tissues of 100 patients with gastric cancer and from four gastric cancer cell lines. Expression of miR-137 was detected by real-time PCR from 100 patients. The effects of miR-137 overexpression on gastric cancer cells’ proliferation, apoptosis, migration and invasion ability were investigated in vitro and in vivo. The target gene of miR-137 was predicted by Targetscan on line software, screened by dual luciferase reporter gene assay and demonstrated by western blot. As a result, the expression of miR-137 was significant reduced in gastric cancer cell line HGC-27, HGC-803, SGC-7901 and MKN-45 as well as in gastric cancer tissues compared with GES-1 cell or matched adjacent non-neoplastic tissues (p<0.001). The re-introduction of miR-137 into gastric cancer cells was able to inhibit cell proliferation, migration and invasion. The in vivo experiments demonstrated that the miR-137 overexpression can reduce the gastric cancer cell proliferation and metastasis. Bioinformatic and western blot analysis indicated that the miR-137 acted as tumor suppressor roles on gastric cancer cells through targeting AKT2 and further affecting the Bad and GSK-3β. In conclusion, the miR-137 which is frequently down-regulated in gastric cancer is potentially involved in gastric cancer tumorigenesis and metastasis by regulating AKT2 related signal pathways.     

人OC-3-VGH卵巢癌细胞裸小鼠肿瘤模型的建立

目的建立人OC-3-VGH细胞株卵巢癌裸小鼠模型并观察该肿瘤生物学生长特性。方法OC-3-VGH细胞株复苏后加入10 mL RPMI-1640培养液,放入培养箱,传2～3代后,取细胞悬液,均以4×106个细胞,每只0.2 mL分别接种至BALB/c雌性裸小鼠皮下,2月后处死取材,观察肿瘤生长特性和转移情况。结果皮下接种一周后,裸鼠长出肿瘤,并随时间而增大,体积呈指数增长,第42天始,明显增大（P〈0.05）。组织学检查发现裸鼠皮下肿瘤细胞均细胞体积较大,细胞核大而染色深,核分裂相较多、异型性明显,接种2个月时,未发生其他组织转移。结论建立了新的卵巢癌动物模型,并初步研究了其生物学特性,为卵巢癌治疗方法的研究拓宽了道路。     

MicroRNA-155 Regulates Cell Survival,Growth, and Chemosensitivity by Targeting FOXO3a in Breast Cancer

Breast cancer is the second leading cause of cancer death in women. Despite improvement in treatment over the past few decades, there is an urgent need for development of targeted therapies. miR-155 (microRNA-155) is frequently up-regulated in breast cancer. In this study, we demonstrate the critical role of miR-155 in regulation of cell survival and chemosensitivity through down-regulation of FOXO3a in breast cancer. Ectopic expression of miR-155 induces cell survival and chemoresistance to multiple agents, whereas knockdown of miR-155 renders cells to apoptosis and enhances chemosensitivity. Further, we identified FOXO3a as a direct target of miR-155. Sustained overexpression of miR-155 resulted in repression of FOXO3a protein without changing mRNA levels, and knockdown of miR-155 increases FOXO3a. Introduction of FOXO3a cDNA lacking the 3′-untranslated region abrogates miR-155-induced cell survival and chemoresistance. Finally, inverse correlation between miR-155 and FOXO3a levels were observed in a panel of breast cancer cell lines and tumors. In conclusion, our study reveals a molecular link between miR-155 and FOXO3a and presents evidence that miR-155 is a critical therapeutic target in breast cancer.     

人脐带间充质干细胞抑制卵巢癌的初步研究

目的:从足月剖腹产分娩新生儿脐带中分离出人脐带间充质干细胞(UC-MSCs),探讨其在体外促进SKOV3卵巢癌细胞凋亡,抑制其增殖的作用。方法:新鲜人脐带洗净后剥离动静脉及脐带外膜,得到脐带Wharton's胶。采用组织块贴壁法分离、纯化得到UC-MSCs细胞,光镜下观察UC-MSCs细胞的形态及贴壁生长情况。收集UC-MSCs细胞培养上清,加入SKOV3细胞共培养后,观察不同作用时间(12 h,24 h,36 h,48 h,60 h,72 h)其体外促进SKOV3卵巢癌细胞凋亡,抑制其增殖的作用。结果:光镜下UC-MSCs细胞成长梭状,单核,并成放射或漩涡状排列。PI染色提示,随着UC-MSCs细胞培养上清对SKOV3卵巢癌细胞作用时间的增加,其发生凋亡的细胞数量增多,且具有统计学意义(P0.05)。MTT实验提示SKOV3细胞增殖活力随UC-MSCs细胞培养上清作用时间的增加而显著下降(P0.05),共培养24 h,48 h,72 h的抑制率分别为17.08%,35.36%,46.83%。结论:UC-MSCs在体外具有明显促进SKOV3卵巢癌细胞凋亡,抑制其增殖的作用。     

Genistein Inhibits Prostate Cancer Cell Growth by Targeting miR-34a and Oncogenic HOTAIR

Objective

Genistein is a soy isoflavone that has antitumor activity both in vitro and in vivo. It has been shown that genistein inhibits many type of cancers including prostate cancer (PCa) by regulating several cell signaling pathways and microRNAs (miRNAs). Recent studies suggest that the long non-coding RNAs (lncRNAs) are also involved in many cellular processes. At present there are no reports about the relationship between gensitein, miRNAs and lncRNAs. In this study, we focused on miRNAs, lncRNA that are regulated by genistein and investigated their functional role in PCa.

Method

Microarray (SurePrint G3 Human GE 8×60K) was used for expression profiling of genistein treated and control PCa cells (PC3 and DU145). Functional assay (cell proliferation, migration, invasion, apoptosis and cell cycle assays) were performed with the PCa cell lines, PC3 and DU145. Both in vitro and in vivo (nude mouse) models were used for growth assays. Luciferase reporter assays were used for binding of miR-34a to HOTAIR.

Results

LncRNA profiling showed that HOTAIR was highly regulated by genistein and its expression was higher in castration-resistant PCa cell lines than in normal prostate cells. Knockdown (siRNA) of HOTAIR decreased PCa cell proliferation, migration and invasion and induced apoptosis and cell cycle arrest. miR-34a was also up-regulated by genistein and may directly target HOTAIR in both PC3 and DU145 PCa cells.

Conclusions

Our results indicated that genistein inhibited PCa cell growth through down-regulation of oncogenic HOTAIR that is also targeted by tumor suppressor miR-34a. These findings enhance understanding of how genistein regulates lncRNA HOTAIR and miR-34a in PCa.     

MicroRNA MiR-214 Regulates Ovarian Cancer Cell Stemness by Targeting p53/Nanog

Previous studies have shown aberrant expression of miR-214 in human malignancy. Elevated miR-214 is associated with chemoresistance and metastasis. In this study, we identified miR-214 regulation of ovarian cancer stem cell (OCSC) properties by targeting p53/Nanog axis. Enforcing expression of miR-214 increases, whereas knockdown of miR-214 decreases, OCSC population and self-renewal as well as the Nanog level preferentially in wild-type p53 cell lines. Furthermore, we found that p53 is directly repressed by miR-214 and that miR-214 regulates Nanog through p53. Expression of p53 abrogated miR-214-induced OCSC properties. These data suggest the critical role of miR-214 in OCSC via regulation of the p53-Nanog axis and miR-214 as a therapeutic target for ovarian cancer.     

MiR-124 Radiosensitizes Human Colorectal Cancer Cells by Targeting PRRX1

One of the challenges in the treatment of colorectal cancer patients is that these tumors show resistance to radiation. MicroRNAs (miRNAs) are involved in essential biological activities, including chemoresistance and radioresistance. Several research studies have indicated that miRNA played an important role in sensitizing cellular response to ionizing radiation (IR). In this study, we found that miR-124 was significantly down-regulated both in CRC-derived cell lines and clinical CRC samples compared with adjacent non-tumor colorectal tissues, MiR-124 could sensitize human colorectal cancer cells to IR in vitro and in vivo. We identified PRRX1, a new EMT inducer and stemness regulator as a novel direct target of miR-124 by using target prediction algorithms and luciferase assay. PRRX1 knockdown could sensitize CRC cells to IR similar to the effects caused by miR-124. Overexpression of PRRX1 in stably overexpressed-miR-124 cell lines could rescue the effects of radiosensitivity enhancement brought by miR-124. Taking these observations into consideration, we illustrated that miR-124 could increase the radiosensitivity of CRC cells by blocking the expression of PRRX1, which indicated miR-124 could act as a great therapeutic target for CRC patients.     

Targeting Ferroptosis by Ubiquitin System Enzymes: A Potential Therapeutic Strategy in Cancer

Ferroptosis is a novel type of regulated cell death driven by the excessive accumulation of iron-dependent lipid peroxidation. Therapy-resistant tumor cells, particularly those in the mesenchymal-like state and prone to metastasis, are highly susceptible to ferroptosis, suggesting that induction of ferroptosis in tumor cells is a promising strategy for cancer therapy. Although ferroptosis is regulated at various levels, ubiquitination is key to post-translational regulation of ferroptotic cell death. E3 ubiquitin ligases (E3s) and deubiquitinating enzymes (DUBs) are the most remarkable ubiquitin system enzymes, whose dysregulation accounts for the progression of multiple cancers. E3s are involved in the attachment of ubiquitin to substrates for their degradation, and this process is reversed by DUBs. Accumulating evidence has highlighted the important role of ubiquitin system enzymes in regulating the sensitivity of ferroptosis. Herein, we will portray the regulatory networks of ferroptosis mediated by E3s or DUBs and discuss opportunities and challenges for incorporating this regulation into cancer therapy.     

Effects of Pharmacological Concentrations of Estrogens on Growth of 3AO Human Ovarian Cancer Cells

During the past two decades, the knowledge of the molecular mechanism by which estrogens exert various functions in different tissues and organs has evolved rapidly. Recent reports demonstrated that estrogen could decrease the cell growth in several types of cancer cells, including ovarian cancer cells. Though experiments explored the possible mechanism of the inhibitory effect, the exact mechanism is responsible for the effect, which remains unclear. The ovary is the main source of the estrogen, estrogen receptor is expressed in several ovarian cell types, including ovarian surface epithelium, the tissue of origin of approximately 90% of the ovarian cancers. It was of great interest to analyze the effects of 17β-estradiol (E2) on apoptosis of ovarian cancer cells, and the identification of E2-regulated specific genes involved in epithelial proliferation apoptosis, thus may be a clue for understanding the progression of ovarian cancer and for the design of new target therapies. To elucidate the mechanism involved, effects of pharmacological concentrations of estrogen were studied in human ovarian cancer cell line 3AO cells. Inhibition of cellular growth of 3AO cells was seen with E2 at concentrations higher than 0.1 μmol/L. The estrogen receptor inhibitor ICI 182780 cannot block the inhibitory effect of E2. It was surprising to find that ICI 182780 itself can inhibit the growth of 3AO cells, and had a collaborative effect with E2. The decreased cell growth induced by E2 was shown to be apoptosis as analyzed by flow cytometry. ERβ was detected in the 3AO ovarian cancer cell line but not ERα. The expression of ERβ was weak, which may partially explain why high but not low dose of E2 needed to induce the apoptosis of 3AO cells. We also observed that membrane impermeable E2, E2-BSA have lost growth inhibitory on 3AO cells, which excluded the membrane effect of E2 as previously reported by many investigators. The p38 kinase inhibitor, SB203580 were partially protected 3AO cells against growth inhibition by E2, while inhibitor of JNK, SP600125 enhanced cell death induced by E2. These results showed that MAPK is implicated in cellular processes involving apoptosis.     

药物治疗浓度的雌激素对卵巢癌3AO细胞生长的影响

在过去的20年里,人们对于雌激素在不同组织、器官中发挥不同功能的分子机制进行了深入的研究,并取得了非常快的进展。最近的研究表明,雌激素能够抑制包括卵巢癌在内的多种肿瘤细胞的生长。卵巢是女性雌激素的主要来源,多种卵巢细胞表达雌激素受体,其中包括90％以上的卵巢癌起源的卵巢表面上皮细胞。雌激素诱导卵巢癌细胞凋亡的研究非常有实验及临床价值,雌激素调控的凋亡相关的特异性基因的发现对于揭示卵巢癌的发生发展以及针对卵巢癌特异性治疗的研究将会提供巨大的帮助。以人卵巢癌3AO细胞为模型,探讨了药物治疗浓度的雌激素对卵巢癌细胞的凋亡诱导作用以及其可能机制。首先用MTT检测方法观察了雌二醇及其受体拮抗剂ICI 182780对3AO细胞生长的影响。研究发现,高于0．1pmol／L浓度的雌二醇能够抑制3AO细胞的生长,其中5pmol／L浓度的雌二醇处理3AO细胞72h后,对3AO细胞的抑制率达到70％。雌激素受体的拮抗剂ICI 182780不但不能阻断雌激素的效应,它本身也能抑制3AO细胞的生长,并且与雌激素有协同效应,并且经流式细胞术证实雌激素及其受体拮抗剂引起的3AO细胞的死亡为凋亡。雌激素对生长的调控是细胞类型特异性的,其机制可能与细胞内雌激素受体不同亚型的表达有关。细胞内雌激素受体β亚型的表达利于细胞凋亡的发生,细胞内雌激素受体α亚型的表达则会保护细胞免于凋亡的发生。在对雌激素诱导的凋亡发生机制的探讨过程中,我们发现3AO细胞只表达雌激素受体β亚型,而不表达雌激素受体α亚型,并且与α亚型相比,β亚型的表达明显降低,这可以解释为何需要高浓度的雌激素才能够诱导3AO细胞凋亡。我们又观察了大分子BSA标记的雌激素对3AO细胞生长的影响,结果发现这种不能通过细胞膜的雌激素失去了对3AO细胞生长的抑制作用,从而排除了雌激素的膜效应。近来的研究表明,MAPK信号通路在调控细胞的生长过程中发挥了重要的作用,并且参与了雌激素调控细胞生长的过程。接下来我们观察了MAPK信号通路在雌激素诱导3AO细胞生长中的作用。研究发现,p38／MAPK激酶的抑制剂SB203580部分的阻断了雌激素的生长抑制效应,而JNK／MAPK激酶的抑制剂SP600125则能促进雌激素的效应,提示MAPK信号通路参与了雌激素的这种效应。