Ji-Ae Shin

Effect of β-phenylethyl isothiocyanate from cruciferous vegetables on growth inhibition and apoptosis of cervical cancer cells through the induction of death receptors 4 and 5.

Cruciferous vegetables have been shown to have the possibility to protect against multistep carcinogenesis. β-Phenylethyl isothiocyanate (PEITC) is one component of these vegetables demonstrated to help fight many types of cancer. The present study examined the apoptotic effects of PEITC and its molecular mechanism in human cervical cancer cell lines (HEp-2 and KB). PEITC induced apoptosis to inhibit cell proliferation. According to the protein chip assay, PEITC increased the expression of the death receptors (DR4 and DR5) and cleaved caspase-3 compared to the DMSO treatment group. PEITC also induced caspase-8 and truncated BID. PEITC down-regulated the phosphorylation of extracellular-related kinase (ERK)1/2, whereas neither phospho-c-Jun NH(2)-terminal kinases (JNK) nor phospho-p38 MAPK was changed. The role of ERK in PEITC-induced apoptosis was also investigated using MEK inhibitor (PD98059). PD98059 increased the expression of DR4 and DR5, activated caspase-3, and cleaved PARP. In addition, PEITC decreased the phosphorylation of MEK. Therefore, the apoptotic mechanism of PEITC in cervical cancer cells involves the induction of DR4 and DR5 through the inactivation of ERK and MEK.

Apoptotic effect of tolfenamic acid in androgen receptor-independent prostate cancer cell and xenograft tumor through specificity protein 1

Tolfenamic acid (Tol) is a non‐steroidal anti‐inflammatory drug that was reported to exhibit anticancer activity in pancreatic and colorectal cancer models. This study examined the role of Tol in the death regulation of PC‐3 and DU145 human androgen‐independent prostate cancer cells. The results showed that Tol inhibited cell growth and induced apoptosis, as evidenced by nuclear fragmentation and cleaved caspase 3 and poly(ADP‐ribose) polymerase. Tol suppressed the specificity protein 1 (Sp1) protein in both PC‐3 and DU145 cells. Tol also attenuated Sp1 mRNA and its promoter activity in DU145 cells, but did not alter them in PC‐3 cells, indicating that Tol degrades Sp1 protein in these cells. Tol also downregulated protein levels, mRNA levels and promoter activities of survivin and myeloid cell leukemia‐1, which are downstream targets of Sp1. The expressions of survivin and Mcl‐1 and cancer cell growth were lower in the PC‐3 cells treated with Sp1 interfering RNA and mithramycin A. Moreover, an oral injection of Tol decreased tumor growth and downregulated the Sp1 protein in athymic nude mice bearing DU145 cell xenografts without hepatotoxicity. Overall, Tol downregulates the Sp1 protein to inhibit growth and induce apoptosis in androgen‐refractory prostate cancers, both in vitro and in vivo, that show resistance against many chemotherapeutic agents. (Cancer Sci 2011; 102: 742–748)

Chemopreventive effect of tolfenamic acid on KB human cervical cancer cells and tumor xenograft by downregulating specificity protein 1.

Earlier studies have shown that tolfenamic acid (Tol) exhibits anticancer activity in several cancer models by inhibiting tumor growth and angiogenesis. However, the chemopreventive effect of Tol on a cervical cancer model and the underlying mechanism of action are unknown. In this study, Tol was found to inhibit cell proliferation by inducing apoptosis without affecting cyclo-oxygenase 2 expression, but ampiroxicam did not. Tol decreases the specificity protein 1 (Sp1) mRNA and its promoter activity in KB cervical cancer cells, and the downregulation of Sp1 protein by affecting several proteins that contain GC-rich sites on their promoters. Studies using small interference RNA and an Sp1-specific inhibitor (mithramycin A) confirmed that the decrease in Sp1 by Tol affects survivin and p27. Tol also inhibited tumor growth and Sp1 protein in athymic nude mice xenografts. These results show that Tol could be a potent anticervical cancer drug that acts by regulating Sp1 protein and its downstream pathways.

Mithramycin A inhibits myeloid cell leukemia-1 to induce apoptosis in oral squamous cell carcinomas and tumor xenograft through activation of Bax and oligomerization.

In several human malignancies, overexpression of myeloid cell leukemia-1 (Mcl-1) confers resistance to induction of apoptosis; however, Mcl-1-mediated inhibition of apoptosis in oral squamous cell carcinoma (OSCC) is not fully understood and has been investigated in this study. The Mcl-1 promoter activators (TPA) and epidermal growth factor (EGF) enhanced neoplastic transformation of JB6 cells and this response was accompanied by enhanced expression of Mcl-1, and knockdown of Mcl-1 by RNA interference (RNAi) decreased JB6 cell transformation. In the same cell line, we also demonstrated that mithramycin A (Mith) decreased TPA-induced JB6 cell transformation and Mcl-1 expression. Mcl-1 was overexpressed in human oral tumors compared with normal oral mucosa and also in several OSCC cell lines including HN22 and HSC-4 cells. Treatment of these cells with Mith also decreased Mcl-1 expression and neoplastic cell transformation, and this was accompanied by induction of several markers of apoptosis. Knockdown of Mcl-1 by RNAi also induced apoptotic cell death. The downregulation of Mcl-1 by Mith and RNAi increased pro-apoptotic protein Bax, resulting in the Bax translocation into mitochondria and its oligomerization. Mith also suppressed tumor growth in vivo and induced apoptosis in tumor by also regulating expression of Mcl-1 and Bax proteins. These indicate a critical role for Mcl-1 in the growth and survival of OSCC and demonstrate that Mith may be a potential anticancer drug candidate for clinical treatment of OSCC.

In vitro apoptotic effects of methanol extracts of Dianthus chinensis and Acalypha australis L. targeting specificity protein 1 in human oral cancer cells

The aims of this study were to evaluate the apoptotic activities and molecular mechanisms of methanol extracts of Dianthus chinensis (MEDC) and Acalypha australis L. (MEAL) in human oral cancer cells.

Apoptotic effect of methanol extract of Picrasma quassioides by regulating specificity protein 1 in human cervical cancer cells.

In the present study, we examined the effects of methanol extracts of Picrasma quassioides (MEPQ) on apoptosis in human cervical cancer cells. The results showed that MEPQ decreased the viability and induced caspase‐dependent apoptosis in HEp‐2 cells. MEPQ decreased specificity protein 1 (Sp1) in HEp‐2 cells, whereas Sp1 mRNA was not changed. We found that MEPQ reduced Sp1 protein through proteasome‐dependent protein degradation, but not the inhibition of protein synthesis. Also, MEPQ increased the expressions of Bad and truncated Bid (t‐Bid) but did not alter other Bcl‐2 family members. The knock‐down of Sp1 by both Sp1 interfering RNA and Mithramycin A, Sp1 specific inhibitor clearly increased Bad and t‐Bid expression to decrease cell viability and induce apoptosis. In addition, MEPQ inhibited cell viability and induced apoptotic cell death through the modulation of Sp1 in KB cells. These results suggest that MEPQ may be a potential anticancer agent for human cervical cancer. Copyright

Apoptotic Effect of Tolfenamic Acid in KB Human Oral Cancer Cells: Possible Involvement of the p38 MAPK Pathway

Nonsteroidal anti-inflammatory drugs (NSAIDs) are known to inhibit cancer growth by inhibiting the activity of cyclooxygenase (COX). However, there is increasing evidence that the COX-independent pathway may be also involved in the inhibitory effect of NSAIDs against tumor progression. Tolfenamic acid is a NSAID that exhibits anticancer activity in pancreatic and colorectal cancer models. In the present study, the anti-tumor effect of tolfenamic acid in KB human oral cancer cells is investigated. The results showed that tolfenamic acid does not alter the expression of the COX proteins, but it inhibits cell growth and induces apoptosis as evidenced by the annexin V positivity, sub-G1 population, nuclear fragmentation and the cleavage of poly ADP-ribose polymerase. In addition, tolfenamic acid also leads to a loss of the mitochondrial membrane potential in KB cells. These effects are related to the activation of p38 mitogen-activated protein kinase (MAPK) pathway. These results suggest that tolfenamic acid-induced apoptotic cell death inhibits cancer growth by activating the p38 MAPK pathway for cancer prevention.

Sulforaphane Increases Cyclin-Dependent Kinase Inhibitor, p21 Protein in Human Oral Carcinoma Cells and Nude Mouse Animal Model to Induce G2/M Cell Cycle Arrest

Previously, our group reported that sulforaphane (SFN), a naturally occurring chemopreventive agent from cruciferous vegetables, effectively inhibits the proliferation of KB and YD-10B human oral squamous carcinoma cells by causing apoptosis. In this study, treatment of 20 and 40 µM of SFN for 12 h caused a cell cycle arrest in the G2/M phase. Cell cycle arrest induced by SFN was associated with a significant increase in the p21 protein level and a decrease in cyclin B expression, but there was no change in the cyclin A protein level. In addition, SFN increased the p21 promoter activity significantly. Furthermore, SFN induced p21 protein expression in a nude mouse xenograft model suggesting that SFN is a potent inducer of the p21 protein in human oral squamous carcinoma cells. These findings show that SFN is a promising candidate for molecular-targeting chemotherapy against human oral squamous cell carcinoma.

Effect of methanol extracts of Cnidium officinale Makino and Capsella bursa-pastoris on the apoptosis of HSC-2 human oral cancer cells

Cnidium officinale Makino and Capsella bursa-pastoris are used as traditional herbs with diverse medicinal effects, including the inhibition of inflammation, reduction of blood pressure and as diuretics, however, the anti-cancer effects of C. officinale Makino and C. bursa-pastoris are poorly defined. The aims of this study were to evaluate the effects of methanol extracts of C. officinale Makino (MECO) and methanol extracts of C. bursa-pastoris (MECB) on the cell growth and apoptosis of HSC-2 human oral cancer cells. MECO and MECB caused growth inhibition and the induction of apoptosis in a concentration-dependent manner in HSC-2 cells. A marked reduction in specificity protein 1 (Sp1) expression following treatment with MECO or MECB was also observed. The downregulation of Sp1 by siRNA resulted in growth inhibition and a reduction of total poly (ADP-ribose) polymerase (PARP) expression. In addition, MECO significantly increased Bax expression levels and MECB increased Bak expression levels and decreased Mcl-1 expression levels. These results suggest that MECO and MECB inhibit cell growth and induce apoptosis via the Sp1 protein, indicating that MECO and MECB are useful bioactive materials and attractive drug candidates for oral cancer.

Chemopreventive effects of synthetic C-substituted diindolylmethanes originating from cruciferous vegetables in human oral cancer cells.

Diindolylmethane (DIM), an isothiocyanate found in cruciferous vegetables, has been shown to have cancer chemopreventive effects. A series of synthetic C-substituted DIMs (C-DIMs) analogs was developed, including DIM-C-pPhtBu and DIM-C-pPhC6H5, which exhibited better inhibitory activity in cancer cells than DIM. This study examined the effects of C-DIMs on the growth of human oral cancer cells. DIM-C-pPhtBu and DIM-C-pPhC6H5 decreased the number of viable KB cells and induced caspase-dependent apoptosis. The apoptotic cell death was accompanied by a change in Bax/Bcl-2 ratio and damage to mitochondrial membrane potential through the induction of death receptor 5 and the cleavage of Bid and caspase 8. Studies on the mechanism of action showed that the apoptotic cell death induced by DIM-C-pPhtBu and DIM-C-pPhC6H5 was mediated by endoplasmic reticulum stress. In addition, C-DIMs inhibited cell proliferation and induced PARP cleavage through death receptor 5 and CHOP in HEp-2 and HN22 cells. This provides the first evidence that synthetic C-DIMs originating from cruciferous vegetables induce apoptosis in human oral cancer cells through the endoplasmic reticulum stress pathway.