Shaker-related Kv1 channels contain four channel-forming α subunits. Subfamily member Kv1.1 often occurs oligomerized with Kv1.2 α subunits in synaptic membranes, and so information was sought on the influence of their positions within tetramers on the channels’ properties. Kv1.1 and 1.2 α genes were tandem linked in various arrangements, followed by expression as single-chain proteins in mammalian cells. As some concatenations reported previously seemed not to reliably position Kv1 subunits in their assemblies, the identity of expressed channels was methodically evaluated. Surface protein, isolated by biotinylation of intact transiently transfected HEK-293 cells, gave Kv1.1/1.2 reactivity on immunoblots with electrophoretic mobilities corresponding to full-length concatenated tetramers. There was no evidence of protein degradation, indicating that concatemers were delivered intact to the plasmalemma. Constructs with like genes adjacent (Kv1.1-1.1-1.2-1.2 or Kv1.2-1.2-1.1-1.1) yielded delayed-rectifying, voltage-dependent K+ currents with activation parameters and inactivation kinetics slightly different from the diagonally positioned genes (Kv1.1-1.2-1.1-1.2 or 1.2–1.1-1.2-1.1). Pore-blocking petidergic toxins, α dendrotoxin, agitoxin-1, tityustoxin-Kα, and kaliotoxin, were unable to distinguish between the adjacent and diagonal concatamers. Unprecedentedly, external application of the pore-blocker tetraethylammonium (TEA) differentially inhibited the adjacent versus diagonal subunit arrangements, with diagonal constructs having enhanced susceptibility. Concatenation did not directly alter the sensitivities of homomeric Kv1.1 or 1.2 channels to TEA or the toxins. TEA inhibition of currents generated by channels made up from dimers (Kv1.1-1.2 and/or Kv1.2-1.1) was similar to the adjacently arranged constructs. These collective findings indicate that assembly of α subunits can be directed by this optimized concatenation, and that subunit arrangement in heteromeric Kv channels affects TEA affinity.

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.

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.