Bioactivity Guided Fractionation and Elucidation of Anti-Cancer Properties of Imperata Cylindrica Leaf Extracts.

BACKGROUND: In our earlier study, we reported the anticancer effect of methanolic extracts of, I. cylindrica leaf (ICL) against human oral squamous cell carcinoma cell lines SCC-9. The cytotoxic effect of ICL methanolic extract was specific to the cancer cells and not to the normal cells. The present study aimed to fractionate the ICL methanolic extract to derive anticancer bioactives. METHODS: The ICL methanolic extract was subjected to a bioactivity guided fractionation. Cytotoxic, cell cycle inhibitory, apoptosis and caspase gene expression inducing activity of the active fractions were evaluated using MTT assay, FACS analysis, Annexin V binding assay and RT-PCR respectively. RESULTS: The hexane fraction of ICL methanolic extract (ICLH) was observed to be the most bioactive fraction. It was shown to possess effective cytotoxic and cell cycle inhibitory activities against SCC-9 cells. The hexane fraction also induced apoptosis in SCC-9 cells which was further established at the level of caspase 3 and 8 gene expressions. CONCLUSION: Overall, the results clearly establish the potential of ICLH extract to inhibit cell proliferation and induce apoptosis in the SCC-9 cells. Further analysis of the ICLH fraction could result in development of effective anticancer therapeutics. The natural abundance of I. cylindrica with its wide geographic distribution could make it a preferred natural resource for obtaining novel, cost-effective, anticancer therapeutics with minimal systemic side effects.

Anti-Cancer Effects of Imperata cylindrica Leaf Extract on Human Oral Squamous Carcinoma Cell Line SCC-9 in Vitro.

Imperata cylindrica, a tall tufted grass which has multiple pharmacological applications is one of the key ingredients in various traditional medicinal formula used in India. Previous reports have shown that I. cylindrica plant extract inhibited cell proliferation and induced apoptosis in various cancer cell lines. To our knowledge, no studies have been published on the effect of I. cylindrica leaf extract on human oral cancers. The present study was undertaken in order to evaluate the anticancer properties of the leaf extract of I. cylindrica using an oral squamous cell carcinoma cell line SCC-9 as an in vitro model system. A methanol extract from dried leaves of I. cylindrica (ICL) was prepared by standard procedures. Effects of the ICL extract on the morphology of SCC-9 cells was visualized by microscopy. Cytotoxicity was determined by MTT assay. Effects of the ICL extract on colony forming ability of SCC-9 cells was evaluated using clonogenic assay. Cell cycle analysis was performed by flow cytometry and induction of apoptosis was determined by DNA fragmentation assay. The ICL extract treatment caused cytotoxicity and induced cell death in vitro in SCC-9 cells in a dose-dependent manner. This treatment also significantly reduced the clonogenic potential and inhibited cell proliferation by arresting the cell cycle in the G2/M phase. Furthermore, DNA fragmentation assays showed that the observed cell death was caused by apoptosis. This is the first report showing the anticancer activity of the methanol extracts from the leaves of I. cylindrica in human oral cancer cell line. Our data indicates that ICL extract could be considered as one of the lead compounds for the formulation of anticancer therapeutic agents to treat/manage human oral cancers. The natural abundance of I. cylindrica and its wide geographic distribution could render it one of the primary resource materials for preparation of anticancer therapeutic agents.

Sodium butyrate modulates pRb phosphorylation and induces cell death in human vestibular schwannomas in vitro.

In the present study, effect of Na-Bu on the pRb phosphorylation was analysed in the primary cultures of 12 VS tumors. Primary cultures of VS tumors were established from the fresh tumor tissues removed surgically and were treated with Na-Bu. Na-Bu treatment for 48 h led to morphological changes and apoptotic cell death in VS tumor cells. Na-Bu treatment decreased level of total pRb and phosphorylated form of pRb and caused specific dephosphorylation at Ser 249/Thr 252 and Ser 567. In the untreated and Na-Bu treated cells (when present), pRb was localised in the nucleus. Moreover, in Na-Bu treated cells the nucleus appeared highly condensed as compared to untreated cells. Results of the present study indicated that Na-Bu treatment modulated pRb phosphorylation status and caused apoptotic cell death in VS tumors.

BAY 61-3606, CDKi, and sodium butyrate treatments alter gene expression in human vestibular schwannomas and cause cell death in vitro.

BACKGROUND: Disrupted kinase and signaling pathways are found in many human cancers and they are implicated in carcinogenesis. Therefore, kinases have been important targets for the development of cancer therapeutics. Human vestibular schwannomas (VS) are the third most common intracranial tumours which occur in the vestibular branch of VIII(th ) cranial nerve. Sodium butyrate (Na-Bu) is a potent histone deacetylase inhibitor (HDACi) and with therapeutic efficacy. Spleen tyrosine kinase (Syk) has been implicated in many immunological consequences and is a putative target for cancer treatment. AIMS AND OBJECTIVES: The present study was undertaken in order to evaluate the effect Na-Bu, 2,4-Diamino-5-oxo-pyrimidine hydrochloride (CDKi), a broad spectrum kinase inhibitor and BAY 61-3606 (Syk inhibitor) on the survival of VS tumour tissues in vitro and their possible effects on cell survival/death and levels of a few key proteins in the treated cells as compared to the untreated cells. MATERIALS AND METHODS: Fresh tumour tissues were collected randomly from 16 patients with sporadic, VS tumours, minced into pieces and maintained in primary cultures. Twenty four hours later these cells were exposed to Na-Bu, BAY 61-3606 or CDKi. Forty eight hours after exposure, the tissue lysates were analysed by western blotting for expression of pRb and other proteins involved in cell survival/death. SUMMARY AND SIGNIFICANCE OF THE FINDINGS: The tissue samples used were positive for S100A protein, the maker for schwann cells confirming the VS tumour samples. The three individual treatments led to morphological change, DNA fragmentation and cell death and significantly reduced level of total and phosphorylated forms of pRb protein and drastically reduced EGF-R protein. These treatments also modulated levels of other proteins involved in cell survival/death such as PI3K, Caspase 3, TGF-ß1, JNK, ASK1, Shh, NF-κB, p21(cip1/waf1). The Untreated cells had uncleaved PARP-1 protein and the treated cells had cleaved PARP-1. The results show that the observed cell death in treated cells perhaps is mediated by modulation of the levels and processing of certain key proteins. The possible development of these components as therapeutics is discussed.

Platelet-derived factors involved in tissue repair-from signal to function.

Topical treatment with platelet derivatives has increasingly been described as being capable of accelerating wound healing and to aid in tissue repair. In vitro data indicate that platelets and their contents have chemotactic, migration-inducing, and mitogenic activities, and a major role of these factors in tissue repair has thus been advocated. However, how platelet-derived factors orchestrate tissue repair at the cellular level remains quite obscure even to those individuals who prescribe platelet derivatives as topical wound healing therapy. The primary objective of this review was to provide the practitioner, inexpert in biochemistry, an overview about signal transduction within cells in response to platelet-derived factors. Concepts from the literature were selected to illustrate how a relatively few units of information can be put together in specific order to allow for complex biologic functions to be elicited. To illustrate how functional complexity emerges from a narrow set of messengers, an analogy between signal transduction and language, or contrapunctual music, has been drawn.

Tyrosine phosphorylation of EGF-R and PDGF-R proteins during acute cutaneous wound healing process in mice.

The effect of topical application of epidermal growth factor (EGF) and platelet-derived growth factors (PDGFs) on the levels of EGF-R and PDGF-R proteins and their tyrosine phosphorylation were analyzed during an acute cutaneous wound healing process in mice. The growth factor-treated wounds had optimum levels of receptor proteins as early as day 1 compared with the control, which had only a basal level. Analysis of the tyrosine phosphorylation of the receptor proteins in control and growth factor-treated wounds indicated that they were phosphorylated until day 5 after wounding. Only the mature forms of alpha-PDGF-R and beta-PDGF-R proteins were phosphorylated and not their precursors. Our results show that rapid attainment of maximum levels of growth factor receptor proteins and their tyrosine phosphorylation as early as day 1 and the maintenance of the same until day 3 appear to aid faster and better wound healing. Topical application of PDGF-AA alone did not facilitate the wound healing process and it also antagonized the EGF-medicated wound healing when applied premixed with EGF or within 30 minutes after EGF application. Under these conditions, the receptor proteins were not phosphorylated. Thus, an increased and sustained level of EGF-R and PDGF-R proteins and their tyrosine phosphorylation appear to accelerate the wound healing process.

Altered structure and deregulated expression of the tumor suppressor gene retinoblastoma (RB1) in human brain tumors.

A total of 40 human brain tumor samples were analyzed for tumor-specific alterations at the RB1 gene locus. Gliomas were more prevalent in younger males and meningiomas in older females. Southern blot analysis revealed loss of heterozygosity (LOH) at the intron 1 locus of RB1 gene in 19.4% of informative cases and this is the first report showing LOH at this locus in human brain tumors. Levels of RB1 mRNA and protein, pRb, and the percentage of hyperphosphorylated form of pRb were also analyzed in these tumors. Normal human fibroblast cell line WI38 was used as control in northern and western analysis. Normal sized RB1 mRNA and protein were present in all the tumor samples. Majority of the gliomas had 2.0-fold or higher levels of RB1 mRNA and most meningiomas had less than 2.0-fold of RB1 mRNA compared to control WI38 cells. The total pRb levels were 2.0-fold or higher in all the tumor samples compared to control. More than 50% of pRb existed in hyperphosphorylated form in all gliomas except two. However, six out of 13 meningiomas had less than 50% of total pRb in the hyperphosphorylated form. These results indicate that the increased percentage of hyperphosphorylated form of pRb in gliomas could provide growth advantage to these tumors. Presence of LOH at the RB1 gene locus and the increased levels of RB1 RNA and protein and increased percentage of hyperphosphorylated form of pRb are indicative of an overall deregulation of pRb pathway in human brain tumors.

Age dependent phosphorylation and deregulation of p53 in human vestibular schwannomas.

Tumor-specific alterations at the p53 gene locus in 30 human vestibular schwannomas (VS) comprising 10 confirmed NF2 cases and 20 sporadic cases were analyzed. We found loss of heterozygosity (LOH) at the first intron of the p53 gene locus in 54% of the informative cases. This is the first report showing LOH at the p53 gene locus in a significant number of human VS and both sporadic and NF2 cases show the LOH event. Increased levels of normal size p53 mRNA and p53 protein were found in all the tumors analyzed. Thus p53 appears to be deregulated in all the tumors suggesting that p53 alterations may be associated with tumor progression in VS. There was a negative significant correlation of patients' age and percentage of Ser 392 phosphorylated p53 protein. The tumor samples obtained from younger patients of 35 yr and below showed higher percentage of Ser 392 phosphorylated p53 protein compared to the tumors of older patients. The increased percentage of Ser 392 phosphorylated p53 protein indicates that it could be involved in the acceleration of tumor growth in the younger patients. Our results suggest that age dependent phosphorylation of p53 protein and deregulation of p53 gene has a role in the development of human vestibular schwannomas.

The effect of epidermal growth factor & platelet-derived growth factors on wound healing process.

BACKGROUND & OBJECTIVES: Various growth factors play significant roles during the process of wound healing. A systematic study was carried out to evaluate the effects of topically applied epidermal growth factor (EGF) and platelet-derived growth factors (PDGF-BB, PDGF-AB, PDGF-AA) in various concentrations on wound healing. Various combinations of these growth factors were also studied to find the best combination for wound healing. METHODS: Wounds were created on a mouse model. Various concentrations and combinations of EGF and PDGFs were applied topically and the effects on wound healing were monitored visually. RESULTS: EGF, PDGF-AB and PDGF-BB when applied alone or in combination, healed the wounds in significantly less time and with less scar tissue formation compared to the controls. The best results were obtained with a combination of EGF (10 ng) and PDGF-BB (15 ng). PDGF-AA even at the maximum concentration of 20 ng did not enhance the wound repair process. A combination of PDGF-AA (20 ng) with EGF (10 ng) did not enhance the wound repair process either. INTERPRETATION & CONCLUSION: Topically applied EGF, PDGF-AB and PDGF-BB enhanced the wound repair process and had a role in decreasing the formation of scar tissue. PDGF-AA either alone or in combination with EGF did not enhance the wound repair process significantly. It appears that PDGF-AA might be limiting the EGF-mediated wound repair and such observation has not been previously reported in studies on wound healing using topically applied growth factors.