Fresh Water Cyanobacteria Geitlerinema sp. CCC728 and Arthrospira sp. CCC729 as an Anticancer Drug Resource.

An increasing number of cancer patients worldwide, especially in third world countries, have raised concern to explore natural drug resources, such as the less explored fresh water filamentous cyanobacteria. Six strains of cyanobacteria (Phormidium sp. CCC727, Geitlerinema sp. CCC728, Arthrospira sp. CCC729, Phormidium sp. CCC731, Phormidium sp. CCC730, and Leptolyngbya sp. CCC732) were isolated (paddy fields and ponds in the Banaras Hindu University, campus) and five strains screened for anticancer potential using human colon adenocarcinoma (HT29) and human kidney adenocarcinoma (A498) cancer cell lines. Geitlerinema sp. CCC728 and Arthrospira sp. CCC729 were the most potent as determined by examination of morphological features and by inhibition of growth by graded concentrations of crude extracts and thin-layer chromatography (TLC) eluates. Cell cycle analysis and multiplex assays using cancer biomarkers also confirmed Geitlerinema sp. CCC728 and Arthrospira sp. CCC729 as cancer drug resources. Apoptotic studies in the cells of A498 (cancer) and MCF-10A (normal human epithelial) exposed to crude extracts and TLC fractions revealed no significant impact on MCF-10A cells emphasizing its importance in the development of anticancer drug. Identification of biomolecules from these extracts are in progress

Effect of <i>Geitlerinema</i> sp. CCC728 crude extract on selected cancer biomarkers.

<p>Multiplex assay showing levels of expression of cancer biomarkers in A498 cancer cells after treatment with crude extract at 100 μg·mL^-1 in the cells showing impact on selected biomarkers.</p

Effect of <i>Arthrospira</i> sp. CCC729 crude extract on selected cancer biomarkers.

<p>Multiplex assay showing levels of expression of cancer biomarkers in A498 cancer cells after treatment with crude extract at 100 μg·mL^-1 in the cells.</p

Apoptotic analysis by Flow cytometry using Annexin V-PI.

<p>A498 as well as MCF-10A cells were exposed to TLC fractions C4 and C5 (<i>Geitlerinema</i> sp. CCC728), D3 and D4 (<i>Arthrospira</i> sp. CCC729). Statistical analysis of A498 cell lines under treatment showed significant impact on cancer cells (F_4,10 = 106.582, p<0.001) <b>(a).</b> As far as MCF-10A cells were concerned they were not significantly affected by fractions selected in experiment (F_4,10 = 2.588, p> 0.05) <b>(b).</b></p

Screening for anticancer activity on NRK52E cell line.

<p>Effects of crude extracts and TLC fractions on normal rat kidney epithelial (NRK52E) cells. <i>Geitlerinema</i> sp. CCC728 crude extract and TLC fractions C2, C3, C4, and C5 (a), <i>Arthrospira</i> sp. CCC729 crude extract and TLC fractions D1, D2, D3, and D4 (b). Significant difference (p<0.001) between pairs, control with crude, C2, C3, C4 and C5, crude with control, C2, C3 and C4. C2 with control, crude and C3 <b>(a),</b> Control with crude, D1, D2, D3 and D4 <b>(b)</b>.</p

Phylogenetic tree of 16S rRNA gene in cyanobacteria generated through BLASTN search in NCBI cyanobacterial database using the neighbor joining algorithm provided by MEGA 5.

<p>Phylogenetic tree of 16S rRNA gene in cyanobacteria generated through BLASTN search in NCBI cyanobacterial database using the neighbor joining algorithm provided by MEGA 5.</p

TLC pattern of cyanobacterial extracts.

<p>TLC patterns of methanolic extracts of lyophilized <i>Geitlerinema</i> sp. CCC728 <b>(a)</b> and <i>Arthrospira</i> sp. CCC729 <b>(c),</b> using CCl4: methanol (9:1). The most potent bands from <i>Geitlerinema</i> sp. CCC728 <b>(b)</b> and <i>Arthrospira</i> sp. CCC729 (d), were purified by a second TLC step using hexane: ethyl acetate (1:1).</p

<i>Arthrospira</i> sp. CCC729 crude extract has no effect on some cancer biomarkers.

<p>Multiplex assay showing levels of expression of cancer biomarkers in A498 cancer cells after treatment with crude extract at 100 μg·mL^-1 in the cells showing no impact on selected biomarkers.</p

<i>Geitlerinema</i> sp. CCC728 crude extract has no effect on some cancer biomarkers.

<p>Multiplex assay showing levels of expression of cancer biomarkers in A498 cancer cells after treatment with crude extract at 100 μg·mL^-1 in the cells showing no impact on selected biomarkers.</p

Cell cycle analysis of A498 cell lines with PI staining using Flow cytometry.

<p>Interaction of cells with most potent fractions from the second TLC purification: control cells without treatment (a), cells <i>vs</i> D3 <b>(b)</b> and D4 <b>(c) from</b><i>Arthrospira</i>, C4 <b>(c)</b> and C5 <b>(d)</b> from <i>Geitlerinema</i>.</p