Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy

Metformin: a potential therapeutic agent for recurrent colon cancer.

Accumulating evidence suggests that metformin, a biguanide class of anti-diabetic drugs, possesses anti-cancer properties. However, most of the studies to evaluate therapeutic efficacy of metformin have been on primary cancer. No information is available whether metformin could be effectively used for recurrent cancer, specifically colorectal cancer (CRC) that affects up to 50% of patients treated by conventional chemotherapies. Although the reasons for recurrence are not fully understood, it is thought to be due to re-emergence of chemotherapy-resistant cancer stem/stem-like cells (CSCs/CSLCs). Therefore, development of non-toxic treatment strategies targeting CSCs would be of significant therapeutic benefit. In the current investigation, we have examined the effectiveness of metformin, in combination with 5-fluorouracil and oxaliplatin (FuOx), the mainstay of colon cancer therapeutics, on survival of chemo-resistant colon cancer cells that are highly enriched in CSCs/CSLCs. Our data show that metformin acts synergistically with FuOx to (a) induce cell death in chemo resistant (CR) HT-29 and HCT-116 colon cancer cells, (b) inhibit colonospheres formation and (c) enhance colonospheres disintegration. In vitro cell culture studies have further demonstrated that the combinatorial treatment inhibits migration of CR colon cancer cells. These changes were associated with increased miRNA 145 and reduction in miRNA 21. Wnt/β-catenin signaling pathway was also down-regulated indicating its pivotal role in regulating the growth of CR colon cancer cells. Data from SCID mice xenograft model of CR HCT-116 and CR HT-29 cells show that the combination of metformin and FuOX is highly effective in inhibiting the growth of colon tumors as evidenced by ∼ 50% inhibition in growth following 5 weeks of combination treatment, when compared with the vehicle treated controls. Our current data suggest that metformin together with conventional chemotherapy could be an effective treatment regimen for recurring colorectal cancer (CRC)

A&B Dose response curves for metformin and/or FuOx in CR HCT-116

<p>(A) and CR HT-29 (B) cells produced by fixed ratio method. Fraction of cells affected by combination of the two drugs (fixed ratio) was higher than either agent alone. Fa represents the fraction of cells affected in response to the treatment. Fa values were used to conduct synergy analysis by CalcuSyn software as described in Materials and Methods. <b>C</b>. Relative survival of CR HT-29 cells in the presence of increasing doses of metformin with and without 2×FuOx. Combination treatment is more effective at all the concentrations. Veh: vehicle alone. Each treatment was performed in quadruplicates, bars represent mean ± standard deviation. * p value<0.05.</p

Tumor growth in metformin/FuOx treated SCID mice injected with CR HCT-116 (left pane) or CR HT-29 cells (right panel) (A).

<p>Bars represent mean ± standard error. * p >0.05. Real time quantitative PCR on RNA extracted from cells isolated from the CR HT-29 tumor (B). CD44 levels decreased and CK20 levels increased in metformin/FuOx treated xenografts. Colonosphere formation in the cells isolated from CR-HT-29 xenografts (C).</p

Quantitative real-time PCR showing the expression of miRNA145 (A) and miRNA21(B) normalized to RNU6B in metformin and/or FuOx treated CR HT-29 cells.

<p>C: metformin/FuOx treatment of CR HCT-116 cells inhibits transcriptional activity of TCF/LEF. D: Western blot analysis shows a reduced expression of β-catenin and c-myc in metformin/FuOx treated CR HCT-116 cells. β-actin was used as loading control. * p<0.05.</p

Combination indices for metformin and FuOx combination therapy, as computed by Calcusyn for CR HCT-116 and CR HT-29 colon cancer cells.

<p>Combination indices for metformin and FuOx combination therapy, as computed by Calcusyn for CR HCT-116 and CR HT-29 colon cancer cells.</p

Boyden chamber analysis of migration of parental and CR HT-29 cells.

<p>Metformin/FuOx treatment reduced the migration of CR HT-29 cells. Each point represents an average of 6 readings. Bars represent mean ± standard deviation (A). Wound healing assay using endothelial (top/red) and CRC (bottom/green). Top left panel: wound at 0 hr; bottom left panel: complete wound healing at 48 hr in CR HT-29 cells; top right panel: partial wound healing in HT-29 cells after 48 hr; bottom right panel: incomplete wound healing in CR HT-29 cells in the presence of 5 mM metformin/FuOx at 48 hr (B). Western blot analysis of metformin/FuOx treated CR H29 cells indicating decreased pAkt levels (C); relative changes in phospho-Akt (pAkt) were calculated as a ratio of pAkt/Akt after normalization to β-actin, which was used as loading control. *p<0.05.</p