Up-regulation Of p21WAF1 Expression Research Articles

Suppression of Metastatic Melanoma Growth in Lung by Modulated Electro-Hyperthermia Monitored by a Minimally Invasive Heat Stress Testing Approach in Mice.

Simple SummaryThe lung is the most frequent site of distant melanoma metastases. Metastases of melanoma in the lungs offer a very poor prognosis, with a 5-year survival rate of below 10%. Hyperthermic therapies including modulated electro-hyperthermia (mEHT) in clinical settings have been used to improve the efficacy of radiotherapy, chemotherapy, and immunotherapy of tumors. In this study, we focused primarily on the optimization of mEHT for targeted lung treatment of mice lungs burdened with B16F10 melanoma pulmonary metastases, with a particular focus on elucidating the mechanism of action of mEHT on treated melanoma cells while investigating any potential treatment-related side effects on normal lung tissue. mEHT showed evidence of significant anti-tumor effects as demonstrated by the reduced number of pulmonary metastatic nodules, DNA damage response, downregulation of Ki67 expression, higher immune cell infiltration, and upregulation of p21waf1 expression in mEHT-treated tumors.Modulated electro-hyperthermia (mEHT) is a novel complementary therapy in oncology which is based on the higher conductivity and permittivity of cancerous tissues due to their enhanced glycolytic activity and ionic content compared to healthy normal tissues. We aimed to evaluate the potential of mEHT, inducing local hyperthermia, in the treatment of pulmonary metastatic melanoma. Our primary objective was the optimization of mEHT for targeted lung treatment as well as to identify the mechanism of its potential anti-tumor effect in the B16F10 mouse melanoma pulmonary metastases model while investigating the potential treatment-related side effects of mEHT on normal lung tissue. Repeated treatment of tumor-bearing lungs with mEHT induced significant anti-tumor effects as demonstrated by the lower number of tumor nodules and the downregulation of Ki67 expression in treated tumor cells. mEHT treatment provoked significant DNA double-strand breaks indicated by the increased expression of phosphorylated H2AX protein in treated tumors, although treatment-induced elevation of cleaved/activated caspase-3 expression was insignificant, suggesting the minimal role of apoptosis in this process. The mEHT-related significant increase in p21waf1 positive tumor cells suggested that p21waf1-mediated cell cycle arrest plays an important role in the anti-tumor effect of mEHT on melanoma metastases. Significantly increased CD3+, CD8+ T-lymphocytes, and F4/80+CD11b+ macrophage density in the whole lung and tumor of treated animals emphasizes the mobilizing capability of mEHT on immune cells. In conclusion, mEHT can reduce the growth potential of melanoma, thus offering itself as a complementary therapeutic option to chemo- and/or radiotherapy.

Antitumor Effects of Fucoidan on Human Colon Cancer Cells via Activation of Akt Signaling.

We identified a novel Akt signaling mechanism that mediates fucoidan-induced suppression of human colon cancer cell (HT29) proliferation and anticancer effects. Fucoidan treatment significantly inhibited growth, induced G1-phase-associated upregulation of p21WAF1 expression, and suppressed cyclin and cyclin-dependent kinase expression in HT29 colon cancer cells. Additionally, fucoidan treatment activated the Akt signaling pathway, which was inhibited by treatment with an Akt inhibitor. The inhibition of Akt activation reversed the fucoidan-induced decrease in cell proliferation, the induction of G1-phase-associated p21WAF1 expression, and the reduction in cell cycle regulatory protein expression. Intraperitoneal injection of fucoidan reduced tumor volume; this enhanced antitumor efficacy was associated with induction of apoptosis and decreased angiogenesis. These data suggest that the activation of Akt signaling is involved in the growth inhibition of colon cancer cells treated with fucoidan. Thus, fucoidan may serve as a potential therapeutic agent for colon cancer.

AKT signaling is involved in fucoidan-induced inhibition of growth and migration of human bladder cancer cells

We identified a novel mechanism of AKT signaling in the fucoidan-induced proliferation and migration of human urinary 5637 cancer cells. Fucoidan treatment showed a significant growth inhibition followed by G1-phase-associated up-regulation of p21WAF1 expression and suppression of cyclins and CDK expression in 5637 cells. Also, fucoidan treatment induced the activation of AKT signaling, which was inhibited by treatment with wortmannin, a PI3K-specific inhibitor. Blockade of the AKT function reversed the fucoidan-mediated inhibition of cell proliferation, the increased G1-phase-associated p21WAF1 expression, and the reduction of cell-cycle proteins. Moreover, treatment with fucoidan blocked migration and invasion of 5637 cells. This inhibition was attributed to decreased expression of MMP-9, which was mediated by down-regulation of AP-1 and NF-κB binding activity. Furthermore, wortmannin treatment abolished the decreased cell migration and invasion and the inhibition of MMP-9 expression via the suppression of NF-κB and AP-1 in fucoidan-treated cells. Similar results were observed in another bladder cancer T-24 cells treated with fucoidan. Finally, overexpression of the AKT gene inhibited the proliferation, migration and invasion of bladder cancer cells. These data suggest that the activation of AKT signaling is involved in growth inhibition and suppression of the migration and invasion of bladder cancer cells treated with fucoidan.

Cordycepin causes p21WAF1-mediated G2/M cell-cycle arrest by regulating c-Jun N-terminal kinase activation in human bladder cancer cells

Cordycepin (3′-deoxyadenosine), a bioactive compound of Cordyceps militaris, has many pharmacological activities. The present study reveals novel molecular mechanisms for the anti-tumor effects of cordycepin in two different bladder cancer cell lines, 5637 and T-24 cells. Cordycepin treatment, at a dose of 200 μM (IC 50) during cell-cycle progression resulted in significant and dose-dependent growth inhibition, which was largely due to G2/M-phase arrest, and resulted in an up-regulation of p21WAF1 expression, independent of the p53 pathway. Moreover, treatment with cordycepin-induced phosphorylation of JNK (c-Jun N-terminal kinases). Blockade of JNK function using SP6001259 (JNK-specific inhibitor) and small interfering RNA (si-JNK1) rescued cordycepin-dependent p21WAF1 expression, inhibited cell growth, and decreased cell cycle proteins. These results suggest that cordycepin could be an effective treatment for bladder cancer.

Gene Expression Profiling Revealed Survivin as a Target of 3,3′-Diindolylmethane-Induced Cell Growth Inhibition and Apoptosis in Breast Cancer Cells

The phytochemical indole-3-carbinol (I3C), found in cruciferous vegetables, and its major acid-catalyzed reaction product 3,3'-diindolylmethane (DIM) showed anticancer activity mediated by its pleiotropic effects on cell cycle progression, apoptosis, carcinogen bioactivation, and DNA repair. To further elucidate the molecular mechanism(s) by which 3,3'-diindolylmethane exerts its effects on breast cancer cells, we have used microarray gene expression profiling analysis. We found a total of 1,238 genes altered in 3,3'-diindolylmethane-treated cells, among which 550 genes were down-regulated and 688 genes were up-regulated. Clustering analysis showed significant alterations in some genes that are critically involved in the regulation of cell growth, cell cycle, apoptosis, and signal transduction, including down-regulation of survivin. Previous studies have shown that antiapoptotic protein survivin is overexpressed in many human cancers, including breast cancer. However, very little or no information is available regarding the consequence of down-regulation of survivin for cancer therapy. We, therefore, hypothesized that down-regulation of survivin as observed by 3,3'-diindolylmethane could be an important approach for the treatment of breast cancer. We have tested our hypothesis using multiple molecular approaches and found that 3,3'-diindolylmethane inhibited cell growth and induced apoptosis in MDA-MB-231 breast cancer cells by down-regulating survivin, Bcl-2, and cdc25A expression and also caused up-regulation of p21(WAF1) expression, which could be responsible for cell cycle arrest. Down-regulation of survivin by small interfering RNA before 3,3'-diindolylmethane treatment resulted in enhanced cell growth inhibition and apoptosis, whereas overexpression of survivin by cDNA transfection abrogated 3,3'-diindolylmethane-induced cell growth inhibition and apoptosis. These results suggest that targeting survivin by 3,3'-diindolylmethane could be a new and novel approach for the prevention and/or treatment of breast cancer.

Interferon regulatory factor-1 gene abnormality and loss of growth inhibitory effect of interferon-alpha in human hepatoma cell lines.

The effect of IFN-alpha on the growth of 5 hepatoma cell lines and a normal liver-derived cell line were examined. IFN dose-dependently inhibited the growth of cell lines except for HLE and PLC/PRF/5 in which the inhibition only occurred at a high concentration over 10,000 IU/ml. IFN-alpha induced the G1 arrest of these cells according to upregulation of p21WAF-1 expression, which is induced in PLC/PRF/5 and HLE only at a high IFN concentration. The receptor for IFN-alpha was well expressed in all the cell lines. DNA rearrangement of IRF-1 was detected in HLE and PLC/PRF/5 by Southern blotting, while IRF-2 gene was preserved. IFN-induced gene expressions were compared between HCC-T, HCC-M and PLC/PRF/5. RT-PCR demonstrated that the full-length IRF-1 and -2 mRNA was transcribed in all cell lines, but the mRNA amount of former gene in PLC/PRF/5 was less than that in HCC-T and HCC-M, about 1/10 by competitive RT-PCR. The sequence analysis of IRF-1 cDNA was performed and the full-length mRNA transcription was reconfirmed in PLC/PRF/5, but no significant point mutation was detected. These results suggest that IFN-alpha inhibits hepatoma growth by increasing p21WAF-1 expression only when the IRF-1 gene is preserved normally.

Up-Regulation of p21 WAF1 by Phorbol Ester and Calcium in Human Keratinocytes through a Protein Kinase C-Dependent Pathway

Terminal differentiation in a variety of cell types has been associated with p53-independent up-regulation of p21WAF1 p21WAF1 mRNA and protein are expressed at low levels in normal human skin, but overexpression of p21WAF1 has been observed in differentiating keratinocytes in involved psoriatic epidermis and in human squamous cell carcinoma. In this study we investigated by immunohistochemistry and Western blotting whether calcium and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, well characterized differentiation signals, induce p21WAF1 in cultured normal human keratinocytes and whether induction of p21WAF1 in this system depends on protein kinase C activation or functional p53. Phorbol ester induced p21WAF1 expression, which was maximal at 4 to 8 h with reduction back to baseline by 24 to 48 h. In contrast, increasing the extracellular Ca2+ concentration from 70 micromol/L to 1.5 mmol/L resulted in upregulation of p21WAF1 expression with a slower time course, with peak induction at 18 to 24 h. No parallel increase in p53 expression was observed in normal human keratinocytes. Up-regulation of p21WAF1 was also observed in response to phorbol ester in HaCaT cells, which carry homozygous and inactivating mutations for p53. Induction of p21WAF1 by phorbol ester and Ca2+ was inhibited by the specific protein kinase C inhibitor Ro 31-8220. The results demonstrate a differential time course of p21WAF1 protein up-regulation in response to phorbol ester and Ca2+, signals that result in keratinocyte differentiation, and suggest that induction of p21WAF1 in differentiating human keratinocytes occurs through protein kinase C-dependent and p53-independent mechanisms.

Up-regulation of p21WAF1 expression in myeloid cells is activated by the protein kinase C pathway.

Phorbol-12-myristate-13-acetate (PMA) induces p21WAF-1 expression in human myeloid leukaemic HL-60 cells. We show that this induction is specifically mediated by protein kinase C (PKC). In addition, the PKC inhibitor Ro 31-8220 with predominant PKC-alpha isoform specificity almost completely inhibited PMA-induced up-regulation of p21WAF1 in HL-60 cells as well as in the myelomonocytic leukaemic U937 cells. Pretreatment of HL-60 cells with Ro 31-8220 also inhibited PMA-induced activation of c-raf-1, a known PKC alpha target. In the phorbol ester-tolerant HL-60 subline (PET) with PKC-beta isoform deficiency PMA or bryostatin-1 induced p21WAF1 expression, but to a lesser extent than in wild-type HL-60 cells. In PET cells, Ro 31-8220 also inhibited PMA and bryostatin-1-induced up-regulation of p21WAF1 expression. Our findings indicate that at least in HL-60 cells up-regulation of p21WAF-1 is specifically activated by PKC. We suggest that PKC isoforms other than beta, presumably the PKC-alpha isoform, are involved in this process.