Cell Cycle-related Proteins P53 Research Articles

Oridonin impedes breast cancer growth by blocking cells in S phase and inhibiting the PI3K/AKT/mTOR signaling pathway

Breast cancer is one of the most common cancers. Oridonin, a traditional Chinese medicine, is believed to inhibit tumor growth, but its particular effects on breast cancer remain unknown. In this study, we examined oridonin's effects on 4T1, MCF-7, and MDAMB-231 cellular activity using CCK8. Scratch assays were used to detect oridonin's effects on cellular migration. Oridonin's effects on the breast cancer cell cycle were studied using flow cytometry, and expression of cell cycle related proteins p53, CDK2, and p21 was detected using Western blot assays. Metabolomics assays were used to detect changes in small molecule metabolites and metabolic pathways in breast cancer cells after treatment with oridonin. Oridonin's effects on breast cancer growth were also studied using xenograft mice. Metabolomics assays were used to detect changes in metabolites and metabolic pathways in xenograft mouse plasma in a control group, model group, and drug administration group. Experimental results showed that oridonin could significantly inhibit breast cancer growth both in vivo and in vitro. Scratch experiments showed that oridonin could inhibit breast cancer cell migration. Oridonin was also able to arrest cells in S phase by affecting several cell cycle-related proteins, including p53, CDK2, and p21. Metabolomic analysis of 4T1 cells identified a total of 33 differential metabolites, including multiple amino acids (such as l-Glutamic acid, l-Asparagine, l-Histidine, l-Valine, and l-Isoleucine). KEGG pathway enrichment analysis showed significant changes in aminoacyl-tRNA biosynthesis, and in multiple amino acid metabolic pathways. Plasma metabolomic analyses of xenograft mice revealed 28 differentially-expressed metabolites between the different animal model groups, including multiple amino acids. KEGG pathway analysis showed significant alterations in multiple amino acid metabolic pathways in oridonin-treated mice. Additionally, changes in the expression of PI3K, AKT and mTOR proteins, as well as in branched amino acids, suggest that oridonin affects the PI3K/AKT/mTOR signaling pathway by inhibiting the biosynthesis of valine, leucine and isoleucine. Taken together, our results suggest that oridonin has strong anti-tumor activity in vitro and in vivo, and has potential as an adjuvant to breast cancer treatment regimens.

Low-dose staurosporine selectively reverses BCR-ABL-independent IM resistance through PKC-α-mediated G2/M phase arrest in chronic myeloid leukaemia

Imatinib (IM) resistance has become a critical problem for the treatment of patients with relapsed chronic myeloid leukaemia (CML), so novel therapies are in need. Various isotypes of protein kinases C (PKCs) are up-regulated in CML and related with BCR-ABL regulating several signalling pathways that are crucial to malignant cellular transformation. However, it is still unknown whether PKC isotypes play crucial roles in IM resistance. Therefore, we herein used a PKC pan-inhibitor staurosporine (St). To protect normal cells from damage, a proper dose of St was used, at which IM-resistant CML cells were selectively killed in combination with IM but normal cells survived. The IM resistance of CML cells was best reversed by 4 nM St alone, mainly depending on the G2/M phase arrest. Cell cycle-related proteins p21, CDK2, cyclin A and cyclin B were down-regulated. Meanwhile, PKC-α was more significantly decreased than other PKC isotypes at this concentration. The PKC-α-dependent G2/M phase arrest was induced by down-regulation of CDC23, an important regulator of mitotic progression. Low-dose St also reversed IM resistance in vivo. In conclusion, low-dose St selectively increased the sensitivity of IM-resistant CML to IM by arresting cell cycle in the G2/M phase through PKC-α-dependent CDC23 inhibition.

Abstract 2032: Inhibition of signal transducers and activators of transcription 3 in human endometrial cancer, using a novel STAT3 inhibitor.

Abstract OBJECTIVE: Signal transducer and activator of transcription 3 (Stat3) proteins have important roles in cancer cell survival and proliferation. Recent studies show constitutive activation of STAT3 is associated with tumor growth and oncogenic transformation in several human cancers, however, little is known about STAT3 expression in endometrial cancer. The objectives of this study were to examine the expression of the STAT family of proteins in endometrial cancer cell lines and to evaluate the efficacy of HO-3867, a novel STAT3 inhibitor. METHODS: Expression of STAT family proteins, cell cycle and apoptotic proteins were evaluated via western blot on Ishikawa, AN3-CA, RL95-2, HEC1B, HEC-1A, and SK-UT1B human endometrial cancer cell lines. Cell viability and proliferation of these cell lines were analyzed with MTT and nucleo counter assay after treatment with increasing concentrations of HO-3867. Cell cycle analysis and apoptosis were performed using flow cytometry and annexin V staining. An ubiquitination assay was used to examine the inhibition of CDK5 degradation. Finally, STAT3 over-expression or suppression experiments were performed using wild-type STAT3 cDNA. RESULTS: Expression of activated STAT1, 2, 5, and 6 was inconsistent among cell lines tested, while pSTAT3 Ser727 had consistently high expression. HO-3867 decreased cell viability through induced G2/M arrest and apoptosis within 24h of treatment. HO-3867 inhibits pSTAT3 Ser727 with no change in total STAT3. This was associated with an increase in cell cycle related proteins p53 and p21, a decrease in anti-apoptotic proteins Bcl2 and Bcl-XL, as well as increased cleavage of caspases. After 24h of treatment with HO-3867, 55-88% of cells were in apoptosis, as detected by Annexin. The downregulation of Ser727 was, at least in part, due to an accelerated inhibition of ubiquitin-dependent CDK5 degradation, as a positive regulator of STAT3 Ser727. Suppression of STAT3 expression with siRNA significantly reduced cell survival. In addition, we confirmed that over-expression of STAT3 cDNA provides resistance to HO-3867. CONCLUSION: Endometrial cancer cells express high levels of pSTAT3 Ser727. HO-3867 induces cell cycle arrest and apoptosis by targeting pSTAT3 Ser727. Our findings suggest a specific role of serine-phosphorylated STAT3 that is independent of tyrosine phosphorylation. HO-3867 may be a potential adjunct to chemotherapy and radiation in the treatment of endometrial cancer. Citation Format: Brent J. Tierney, Georgia A. McCann, Kellie S. Rath, Meryl Sudhakar, Shan Naidu, Periannan Kuppusamy, David E. Cohn, Karuppaiyah Selvendiran. Inhibition of signal transducers and activators of transcription 3 in human endometrial cancer, using a novel STAT3 inhibitor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2032. doi:10.1158/1538-7445.AM2013-2032

Abstract 2802: Selective inhibition of signal transducers and activators of transcription 3 overexpressing endometrial cancer cells, using HO-3867, a novel STAT3 inhibitor

Abstract OBJECTIVE: Signal transducer and activator of transcription 3 (STAT3) proteins have important roles in cancer cell survival and proliferation. Recent studies show constitutive activation of STAT3 is associated with tumor growth and oncogenic transformation in several human cancers; however, little is known about STAT3 expression in endometrial cancer. The objectives of this study were to examine the expression of the STAT family of proteins in endometrial cancer cell lines and to evaluate the efficacy of HO-3867, a novel STAT3 inhibitor. METHODS: Expression of STAT family proteins, cell cycle and apoptotic proteins were evaluated via western blot on Ishikawa, AN3-CA, RL95-2, HEC1B, HEC-1A, and SK-UT1B human endometrial cancer cell lines. Cell viability and proliferation of these cell lines were analyzed with MTT and nucleo counter assay after treatment with increasing concentrations of HO-3867. Cell cycle analysis and apoptosis were performed using flow cytometry and annexin V staining. An ubiquitination assay was used to examine the inhibition of CDK5 degradation. Finally, STAT3 over-expression or suppression experiments were performed using wild-type STAT3 cDNA and STAT3 siRNA. RESULTS: Expression of activated STAT1, 2, 5, and 6 was inconsistent among cell lines tested, while pSTAT3 Ser727 had consistently high expression. HO-3867 decreased cell viability through induced G2/M arrest and apoptosis within 24h of treatment. HO-3867 inhibited pSTAT3 Ser727 with no change in total STAT3. This was associated with an increase in expression of cell cycle related proteins p53 and p21, a decrease in anti-apoptotic proteins Bcl2 and Bcl-XL, as well as increased cleavage of caspases. After 24h of treatment with HO-3867, 55-88% of cells were apoptotic. The downregulation of Ser727 was, at least in part, due to an accelerated inhibition of ubiquitin-dependent CDK5 degradation, as a positive regulator of STAT3 Ser727. Suppression of STAT3 expression with siRNA significantly reduced cell survival. In addition, we confirmed that over-expression of STAT3 cDNA provides resistance to HO-3867. CONCLUSION: Endometrial cancer cells express high levels of pSTAT3 Ser727. HO-3867 induces cell cycle arrest and apoptosis by targeting pSTAT3 Ser727. Our findings suggest a specific role of serine-phosphorylated STAT3 that is independent of tyrosine phosphorylation. HO-3867 may be a potential adjunct to chemotherapy and radiation in the treatment of endometrial cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2802. doi:1538-7445.AM2012-2802

Immunohistochemical study of the expression of cell cycle regulating proteins at different stages of bladder cancer.

The cell cycle is known to be deregulated in cancer. We therefore analyzed the expression of the cell cycle related proteins p21, p27, p16, Rb, and L-myc by immunohistochemical staining of bladder tumors. The tissue material consisted of bladder tumors from three groups of patients; group 1, 23 patients with recurrent stage Ta (non-invasive) tumors; group 2, 22 patients presenting at their first admission with T2-4 (muscle invasive) tumors; group 3, 24 patients who experienced disease progression from Ta or T1 (invasive in connective tissue) to a higher stage. By immunohistochemical staining the protein expression was compared to allelic deletions of the corresponding genes. The allelic deletions were detected by PCR-based microsatellite analyses. We detected a significant reduction in the expression levels of the cell cycle related proteins p21(waf1) ( P=0.002), p27(kip1) ( P=0.03), Rb ( P=0.00002), and L-myc ( P=0.00000007) in muscle invasive tumors compared to noninvasive tumors. Tumors presenting as muscle invasive at first diagnosis had significantly lower levels of p16/CDKN2A ( P=0.01) when compared to muscle invasive tumors that followed Ta or T1 precursor lesions. We found no general correlation between allelic deletion of a gene and its immunohistochemical protein expression, indicating that the remaining allele may be capable of encoding a normal or even increased protein level. Our results support the hypothesis that bladder tumors with invasion at first diagnosis differ from those in which invasion follow superficial tumors. This difference is reflected as a different level in cell cycle related protein expression. The data also indicate that allelic deletions of cell cycle related genes do not correlate with an altered level of protein expression.