EGR1-dependent PTEN upregulation by 2-benzoyloxycinnamaldehyde attenuates cell invasion and EMT in colon cancer

There has been little evidence to support EGR1 and PTEN function on the EMT of cancer cells. We tried to evaluate how these genes affect cancer cell invasion and EMT through investigating the molecular mechanism(s) of 2′-benzoyloxycinnamaldehyde (BCA). Matrigel invasion and wound healing assay, and in vivo mice model were used to evaluate the effect of BCA on colon cancer cell migration. The molecular mechanism(s) of BCA were evaluated by knock-down or overexpression of EGR1 and PTEN. BCA at 50 nM increased E-cadherin and EGR1 expression without cytotoxicity. Cell migration was inhibited significantly by BCA both in vitro and in vivo. Moreover, BCA inhibits Snail and Vimentin expression, as well as β-catenin nuclear accumulation. Suppression of EGR1 by siRNA attenuated the inhibition of matrigel invasion by BCA, indicating that EGR1 is responsible for BCA effect. PTEN was upregulated by BCA treatment or EGR1 overexpression. In addition, shPTEN transfection stimulated EMT and cell invasion in vitro. Our data suggest that BCA leads to a remarkable upregulation of EGR1 expression, and that EMT and invasion is decreased via EGR1-dependent PTEN activation. These data showed a critical role of EGR1-PTEN signaling pathway in the EMT of colon cancer, as well as metastasis.     

Synergy of 5-aza-2′-deoxycytidine (DAC) and paclitaxel in both androgen-dependent and -independent prostate cancer cell lines

To determine the synergy of 5-aza-2′-deoxycytidine (DAC) and paclitaxel (PTX) against prostate carcinoma (PC) cells by isobolographic analysis. We demonstrated that DAC could significantly increase the susceptibility of PC cells to PTX, and confirmed the synergy of DAC and PTX. DAC enhanced the PTX induced up-regulation of caspase activity and antiproliferative effect, resulting in an increase of cells in subG1 and G2/M phases. In addition, the synergy was observed in both androgen-dependent and -independent PC cell lines. It suggested that combination chemotherapy with DAC and PTX might be a new strategy to improve the clinical response rate of PC.     

2′-Behenoyl-paclitaxel conjugate containing lipid nanoparticles for the treatment of metastatic breast cancer

The aim of these studies was to develop a novel 2′-behenoyl-paclitaxel (C22-PX) conjugate nanoparticle (NP) formulation for the treatment of metastatic breast cancer. A lipophilic paclitaxel derivative C22-PX was synthesized and incorporated into lipid-based NPs. Free C22-PX and its NP formulation were evaluated in a series of in vitro and in vivo studies. The results demonstrated that C22-PX NPs were much better tolerated and had significantly higher plasma and tumor AUCs compared to Taxol at the maximum tolerated dose (MTD) in a subcutaneous 4T1 mouse mammary carcinoma model. These benefits resulted in significantly improved antitumor efficacy with the NP-based formulation.     

β-asarone suppresses HCT116 colon cancer cell proliferation and liver metastasis in part by activating the innate immune system

Studies have revealed that β-asarone exerts a powerful inhibitory effect on the proliferation of human cancer cells. The authors'' previous study demonstrated that β-asarone could induce LoVo colon cancer cell apoptosis in vitro and in vivo, indicating its anticancer properties. The present study aimed to determine the antineoplastic effect of β-asarone in HCT116 colon cancer cells. An in vitro proliferation assay using a real time cell analyzer demonstrated that β-asarone effectively decreased HCT116 cell proliferation in a dose-dependent manner. Bioinformatics analysis revealed that differentially expressed genes following β-asarone inhibition were involved in the ‘cell cycle’, ‘cell division’, ‘cell proliferation’ and ‘apoptosis’. Subsequently, a xenograft assay evidenced the inhibitory effect of β-asarone on the growth of HCT116 tumors in vivo. Further detection of immune-associated cytokines and cells suggested that β-asarone might be involved in the antitumor immune response by stimulating granulocyte-colony stimulating factor and increasing the number of macrophage cells in the spleen. Additionally, a murine model of splenic-transplantation verified the strong suppressive role of β-asarone in colon cancer liver metastasis in vivo. Taken together, the results of the current study revealed that β-asarone decreased HCT116 colon cancer cell proliferation and liver metastasis potentially by activating the innate immune system, supporting the multi-system regulation theory and providing a basis for further mechanistic studies on colon cancer.     

Enhanced cytotoxicity with a novel system combining the paclitaxel-2′-ethylcarbonate prodrug and an HSV amplicon with an attenuated replication-competent virus,HF10 as a helper virus

We previously demonstrated that HF10, which is a natural, non-engineered HSV-1, has potent oncolytic activity in the treatment of solid malignant tumors in vitro and in vivo [H. Takakuwa, F. Goshima, N. Nozawa, T. Yoshikawa, H. Kimata, A. Nakao, et al., Oncolytic viral therapy using a spontaneously generated herpes simplex virus type 1 variant for disseminated peritoneal tumor in immunocompetent mice, Arch. Virol. 148 (2003) 813–825; S. Kohno, C. Lou, F. Goshima, Y. Nishiyama, T. Sata, Y. Ono, Herpes simplex virus type 1 mutant HF10 oncolytic viral therapy for bladder cancer, Urology 66 (2005) 1116–1121; D. Watanabe, F. Goshima, I. Mori, Y. Tamada, Y. Matsumoto, Y. Nishiyama, Oncolytic virotherapy for malignant melanoma with herpes simplex virus type 1 mutant HF10, J. Dermatol. Sci. 50 (2008) 185–196; A. Nawa, C. Luo, L. Zhang, Y. Ushijima, D. Ishida, M. Kamakura, et al., Non-engineered, naturally oncolytic herpes simplex virus HSV1 HF10: applications for cancer gene therapy, Curr. Gene. Ther. 8 (2008) 208–221]. Previous reports have also shown that a combination of HF10 and paclitaxel (TAX) was more efficacious than either regimen alone for some types of malignant tumors [S. Shimoyama, F. Goshima, O. Teshigahara, H. Kasuya, Y. Kodera, A. Nakao, et al., Enhanced efficacy of herpes simplex virus mutant HF10 combined with paclitaxel in peritoneal cancer dissemination models, Hepatogastroenterology 54 (2007) 1038–1042]. In this study, we investigated the efficacy of gene-directed enzyme prodrug therapy (GDEPT) using a novel system that combines the paclitaxel-2′-ethylcarbonate prodrug (TAX-2′-Et) and an HSV amplicon expressing rabbit-carboxylesterase (CES) with HF10 as a helper virus. This GDEPT system aims to produce high level of CES at the tumor site, resulting in efficient local conversion of the TAX-2′-Et prodrug into the active drug TAX [A. Nawa, T. Tanino, C. Lou, M. Iwaki, H. Kajiyama, K. Shibata, et al., Gene directed enzyme prodrug therapy for ovarian cancer: could GDEPT become a promising treatment against ovarian cancer?, Anti-Cancer Agents Med Chem 8 (2008) 232–239].     

Inhibition of Plk1 and Pin1 by 5'-nitro-indirubinoxime suppresses human lung cancer cells

A novel indirubin derivative, 5′-nitro-indirubinoxime (5′-NIO), exhibits a strong anti-cancer activity against human cancer cells. Here, the 5′-NIO-mediated G1 cell cycle arrest in lung cancer cells was associated with a decrease in protein levels of polo-like kinase 1 (Plk1) and peptidyl-prolyl cis/trans isomerase Pin1. Treatment with Plk1 siRNA or Pin1 inhibitor effectively inhibited the Rb phosphorylation, suggesting their regulatory role at G1 phase. In addition, the overexpression of Plk1 or Pin1 inhibited apoptotic signals following the cleavage of PARP in 5′-NIO-treated cells. These findings suggest that 5′-NIO have potential anti-cancer efficacy through the inhibition of Plk1 or/and Pin1 expression.     

Timing is everything: Order of administration of 5-aza 2′ deoxycytidine,trichostatin A and tamoxifen changes estrogen receptor mRNA expression and cell sensitivity

Restoration of estrogen receptor (ER) expression using epigenetic inhibitors re-establishes expression of the estrogen receptor (ER) and restores tamoxifen sensitivity in ER negative breast cancer cells. We tested if order of administration of the DNMT (5-aza 2′ deoxycytidine/AZA) or HDAC (trichostatin A/TSA) inhibitors and tamoxifen affected ER re-expression and tamoxifen sensitivity. Treatment with AZA followed by co-administration of TSA plus tamoxifen resulted in the greatest ER re-expression and tamoxifen sensitivity, although sensitivity was not increased as robustly as expected. This could be due to increased cytoplasmic levels of HuR, suggesting that cytoplasmic HuR levels are central to tamoxifen responsiveness.     

5′-Nitro-indirubinoxime,an indirubin derivative,suppresses metastatic ability of human head and neck cancer cells through the inhibition of Integrin β1/FAK/Akt signaling

Head and neck cancer is a malignant cancer and has the high infiltrative potential leading to metastasis. The objective of the study was to investigate the effects of 5′-nitro-indirubinoxime (5′-NIO), an indirubin derivative, on metastasis of head and neck cancer cells and to explore the underlying molecular mechanisms involved in this process. After treatment of head and neck cancer cells with 5′-NIO, cell metastatic behaviors such as colony formation, invasion, and migration were inhibited in a concentration-dependent manner. 5′-NIO inhibited the beta1 Integrin/FAK/Akt pathway which can then facilitate invasion and/or migration of cancer cells through the extracellular matrix (ECM). Moreover, treatment of head and neck cancer cell with Integrin β1 siRNA or FAK inhibitor effectively inhibited the invasion and migration, suggesting their regulatory role at invasiveness and migratory of head and neck cancer cells. In vivo CAM assay, treatment with 5′-NIO reduced the angiogenesis in FaDu cells xenograft fertilized chicken eggs, primarily by inhibiting expression of VEGF. We conclude that 5′-NIO inhibits the metastatic ability of head and neck cancer cells by blocking the Integrin β1/FAK/Akt pathway.     

Protein‐arginine deiminase 2 suppresses proliferation of colon cancer cells through protein citrullination

Expression of the gene for protein‐arginine deiminase 2 (PADI2) has been shown to be downregulated in colon cancer, with such downregulation being indicative of a poor prognosis in individuals with this disease. We have now examined the expression of PADI2 in matched colon cancer and normal colon tissue specimens as well as in colon cancer cell lines. We found that isoform 1 of PADI2 is the predominant isoform in colon tissue and is downregulated during colon carcinogenesis. Immunohistochemical analysis showed that PADI2 is expressed in normal colonic epithelial cells. Overexpression of PADI2 isoform 1 suppressed the proliferation of colon cancer cells in vitro in association with increased protein citrullination. Expression of a catalytically inactive mutant (C647A) of PADI2 or of PADI2 isoform 2 did not induce such effects, indicating that the protein citrullination activity of PADI2 is required for inhibition of cell growth. The growth defect induced by PADI2 was not attributable to increased apoptosis but rather was accompanied by arrest of cell cycle progression in G₁ phase. Finally, we detected citrullinated proteins in normal colon tissue by immunoblot analysis. Our data thus suggest that PADI2 suppresses the proliferation of colonic epithelial cells through catalysis of protein citrullination, and that downregulation of PADI2 expression might therefore contribute to colon carcinogenesis.     

Dihydroartemisinin synergistically enhances the cytotoxic effects of oxaliplatin in colon cancer by targeting the PHB2-RCHY1 mediated signaling pathway

Dihydroartemisinin (DHA) has recently attracted increasing attention for its low toxicity and high antitumor activity. DHA has been reported to have synergistic anticancer effects with a variety of drugs in the clinic; however, the molecular mechanism by which DHA inhibits tumorigenesis and improves oxaliplatin cytotoxicity in colon cancer cells is still not well understood. In this study, we found that DHA can inhibit cell proliferation and colony formation in a dose-dependent manner. Prohibitin 2 (PHB2) is a potential target by which DHA exerts its antitumor and cytotoxic effects. The function and molecular mechanism of PHB2 in colon cancer tumorigenesis were fully studied to determine the regulatory mechanism between DHA and PHB2. We found that PHB2, a mitochondrial inner membrane scaffold protein, has a higher expression level in colon cancer tissues than in adjacent nontumor tissues and is mainly localized in mitochondria. Overexpression of PHB2 can promote cell proliferation and colony formation in vitro and accelerate tumor growth in vivo. We also found that the expression level of PHB2 was inversely related to the cytotoxicity of DHA and oxaliplatin in colon cancer cells. The molecular mechanism of PHB2 in tumorigenesis and cancer therapy was further studied. The results showed that 20 μM DHA can downregulate PHB2 expression in a ubiquitylation-dependent manner and subsequently block PHB2-induced RCHY1 upregulation and p53 and p21 downregulation. In this process, RCHY1 is necessary for PHB2 to play a tumor-promoting role. Thus, PHB2 and RCHY1 are effective targets for colon cancer therapy, and DHA has synergistic anticancer effects with oxaliplatin via promoting PHB2 degradation in colon cancer cells.     

Effects of trans-(±)-kusunokinin on chemosensitive and chemoresistant ovarian cancer cells

Ovarian cancer ranks eighth in cancer incidence and mortality among women worldwide. Cisplatin-based chemotherapy is commonly used for patients with ovarian cancer. However, the clinical efficacy of cisplatin is limited due to the occurrence of adverse side effects and development of cancer chemoresistance during treatment. Trans-(±)-kusunokinin has been previously reported to inhibit cell proliferation and induce cell apoptosis in various cancer cell types, including breast, colon and cholangiocarcinoma. However, the potential effects of (±)-kusunokinin on ovarian cancer remains unknown. In the present study, chemosensitive ovarian cancer cell line A2780 and chemoresistant ovarian cancer cell lines A2780cis, SKOV-3 and OVCAR-3 were treated with trans-(±)-kusunokinin to investigate its potential effects. MTT, colony formation, apoptosis and multi-caspase assays were used to determine cytotoxicity, the ability of single cells to form colonies, induction of apoptosis and multi-caspase activity, respectively. Moreover, western blot analysis was performed to determine the proteins level of topoisomerase II, cyclin D1, CDK1, Bax and p53-upregulated modulator of apoptosis (PUMA). The results demonstrated that trans-(±)-kusunokinin exhibited the strongest cytotoxicity against A2780cis cells with an IC₅₀ value of 3.4 µM whilst also reducing the colony formation of A2780 and A2780cis cells. Trans-(±)-kusunokinin also induced the cells to undergo apoptosis and increased multi-caspase activity in A2780 and A2780cis cells. This compound significantly downregulated topoisomerase II, cyclin D1 and CDK1 expression, but upregulated Bax and PUMA expression in both A2780 and A2780cis cells. In conclusion, trans-(±)-kusunokinin suppressed ovarian cancer cells through the inhibition of colony formation, cell proliferation and the induction of apoptosis. This pure compound could be a potential targeted therapy for ovarian cancer treatment in the future. However, studies in an animal model and clinical trial need to be performed to support the efficacy and safety of this new treatment.     

Epigenetic repolarization of T lymphocytes from chronic lymphocytic leukemia patients using 5-aza-2'-deoxycytidine

T cell immune dysfunction has an important role in the profound immunosupression that characterizes chronic lymphocytic leukemia (CLL). Improper polarization of T cells has been proposed as one of the mechanism involved. Mounting data implicates chromatin regulation, namely promoter methylation, in the plasticity of naïve human T cells. Recent in vitro evidence indicates that this plasticity may be phenotypically altered by using methylation inhibitors which are approved for clinical use in certain types of cancer. These results beg the question: can the ineffective polarization of T lymphocytes in the context of CLL be effectively modulated using methylation inhibitors in a sustainable therapeutic fashion? To answer this question our laboratory has studied the effects of 5-aza-2′-deoxycytidine (5A2) in helper and cytotoxic T lymphocytes from healthy donors and CLL patients in well characterized molecular and epigenetic signaling pathways involved in effective polarization. Moreover, we sought to investigate the consequences of methylation inhibitor treatment on lymphocyte survival, activation intensity, and naïve cell polarization. Our data indicates that 5A2 treatment can repolarize Th2 cells to effectively secrete interferon gamma, signal via T-bet, and achieve demethylation of critical Th1 specific promoters. Moreover, we demonstrate that 5A2 can force Th1 polarization of naïve T cells despite a strong IL-4 stimuli and a lack of IL-12. In conclusion our data seeks to define a modality in which improper or ineffective T cell polarization can be altered by 5AZA and could be incorporated in future therapeutic interventions.     

Activation of β2‐adrenergic receptor signals suppresses mesenchymal phenotypes of oral squamous cell carcinoma cells

Metastasis is a primary reason related to the mortality of oral squamous cell carcinoma (OSCC) patients. A program called epithelial‐mesenchymal transition (EMT) has been shown to play a critical role in promoting metastasis in epithelium‐derived carcinoma. During EMT, epithelial cancer cells acquire motile mesenchymal phenotypes and detach from primary tumors. Recent lines of evidence have suggested that EMT confers cancer cells with tumor‐initiating ability. Therefore, selective targeting of EMT would lead to the development of effective therapeutic agents. In this study, using a chemical biology approach, we identified isoxsuprine, a β2‐adrenergic receptor (β2‐AR) agonist as a low‐molecular‐weight compound that interferes with the acquisition of mesenchymal phenotypes of oral cancer cells. Treatment of multiple types of oral cancer cells with isoxsuprine led to the downregulation of mesenchymal cell markers that was accompanied by reduced cell motility. Similar inhibitory effects were also observed for isoprenaline, a non‐selective β‐adrenergic receptor (β‐AR) agonist. In addition, inhibition of cell migration upon treatment with isoxsuprine was reverted by a non‐selective β‐AR antagonist, propranolol, and the CRISPR/Cas9 system‐mediated deletion of the β2‐AR gene, suggesting that the effects exerted by isoxsuprine involved signals mediated by β2‐AR. In addition, in a subcutaneous xenograft model of oral cancer cells, the administration of isoxsuprine effectively suppressed primary tumor growth, suggesting β2‐AR signals to be a promising cancer therapeutic target for treatment of OSCC.     

Potentiation of cytotoxicity and radiosensitization of (E)‐2‐deoxy‐2′‐(fluoromethylene) cytidine by pentoxifylline In vitro

(E)‐2′‐deoxy‐2′‐(fluoromethylene) cytidine (FMdC), a novel inhibitor of ribonucleotide‐diphosphate reductase, has been shown to have anti‐tumor activity against solid tumors and sensitize tumor cells to ionizing radiation. Pentoxifylline (PTX) can potentiate the cell killing induced by DNA‐damaging agents through abrogation of DNA‐damage‐dependent G₂ checkpoint. We investigated the cytotoxic, radiosensitizing and cell‐cycle effects of FMdC and PTX in a human colon‐cancer cell line WiDr. PTX at 0.25–1.0 mM enhanced the cytotoxicity of FMdC and lowered the IC₅₀ of FMdC from 79 ± 0.1 to 31.2 ± 2.1 nM, as determined by MTT assay. Using clonogenic assay, pre‐irradiation exposure of exponentially growing WiDr cells to 30 nM FMdC for 48 hr or post‐irradiation to 0.5 to 1.0 mM PTX alone resulted in an increase in radiation‐induced cytotoxicity. Moreover, there was a significant change of the radiosensitization if both drugs were combined as compared with the effect of either drug alone. Cell‐cycle analysis showed that treatment with nanomolar FMdC resulted in S‐phase accumulation and that such an S‐phase arrest can be abrogated by PTX. Treatment with FMdC prior to radiation increased post‐irradiation‐induced G₂ arrest, and such G₂ accumulation was also abrogated by PTX. These results suggest that pharmacological abrogation of S and G₂ checkpoints by PTX may provide an effective strategy for enhancing the cytotoxic and radiosensitizing effects of FMdC. Int. J. Cancer 80:155–160, 1999. © 1999 Wiley‐Liss, Inc.     

3-Deazauridine enhances the antileukemic action of 5-aza-2′-deoxycytidine and targets drug-resistance due to deficiency in deoxycytidine kinase

New approaches should be sought to treat high-risk acute lymphoblastic leukemia (ALL). Since aberrant DNA methylation plays an important role in leukemogenesis of ALL, it can be targeted by 5-aza-2′-deoxycytidine (5-AZA-CdR), a potent inhibitor of DNA methylation. 5-AZA-CdR is a prodrug that is activated by deoxycytidine kinase (DCK). Leukemic cells lacking DCK are drug-resistant. In a previous phase I study, we reported that 5-AZA-CdR could induce remissions in ALL. However, some patients developed drug-resistance due to deficiency in DCK. These observations aroused our interest in 3-deazauridine (3-DU), a CTP synthetase inhibitor that is effective against leukemic cells deficient in DCK. In this report, we observed that 3-DU enhanced the in vitro antineoplastic action of 5-AZA-CdR on human leukemic cells by increasing its incorporation into DNA. Using an optimized dose-schedule we showed that this combination could cure some mice bearing L1210 leukemia, even in the presence of a subpopulation of drug-resistant (L1210/ARA-C) leukemic cells lacking DCK. 3-DU alone also cured some mice with L1210/ARA-C leukemia. In a pilot study on 3 relapsed patients with advanced ALL, the combination of 5-AZA-CdR and 3-DU produced a marked reduction in leukemic blasts, confirming our preclinical observations. Furthermore, after several treatments with these agents all three patients developed drug-resistance to 5-AZA-CdR as determined by an in vitro drug sensitivity test. In two patients we showed by enzymatic analysis that the drug-resistance was due to deficiency in DCK. Our preclinical and clinical results provide a strong rationale to further investigate the combination of 5-AZA-CdR and 3-DU for the treatment of advanced ALL.     

转录因子Oct4、Sox2在结肠癌中的表达及意义

目的:研究转录因子Sox2、Oct4在结肠癌中的表达及相互关系,探索两者参与结肠癌发生发展中的临床意义。方法:用免疫组织化学SP法检测Oct4、Sox2在50例结肠癌及其匹配的癌旁组织中的表达。Western blot检测其在结肠癌细胞株SW480、SW620、Lovo及永生化结肠上皮细胞株HIEC中的表达。结果:免疫组织化学结果显示Oct4、Sox2在结肠癌组织中阳性表达率分别为80%(40/50),74%(37/50),显著高于匹配的癌旁组织,后者表达率分别为48%(24/50),20%(10/50)(P<0.05),并且两者阳性表达率呈正相关关系(r=0.465,P<0.05)。Oct4表达与病理级别呈负相关,而与年龄、性别无相关性。Sox2表达与患者的年龄、性别、病理级别无明显相关性。Western blot显示结肠癌细胞株与永生化结肠上皮细胞HIEC均表达Oct4,Sox2;Oct4、Sox2在结肠癌高转移细胞系SW620较亲本细胞SW480表达增高。结论:转录因子Oct4、Sox2在结肠癌组织中表达高于癌旁组织,并且两者的表达存在正相关性。提示Oct4和Sox2在结肠癌的发生中起着重要的作用。