Figure 6. Ohmline as a new personalized treatment strategy to decrease P-Akt and therefore modulate the effects of Anti-EGFR mAbs.

A. Disrupting lipid-rafts with the alkyl-lipid Ohmline allows SK3 to re-translocate outside away from lipid-rafts without modifying the localization of calcium channels whereas the co-treatment Ohmline/Tg the translocation of calcium channels into lipid-raft. Immunoblots representing membrane fractionation, on a sucrose gradient, of cells treated with Ohmline alone (middle panel) or associated with 5μM Tg for 20 min (right panel). B. Left panel, Dissociation of the lipid-raft Orai1/TRPC1/SK3 channel complex by Ohmline decreased Ca²⁺entry evoked by Tg. Fluorescence measurement and relative fluorescence of Ca²⁺entry after intracellular calcium store depletion by Tg in cells treated 24h with Ohmline. Data represent means ± SEM. *p<0.05, sample significantly different from control (N=4, Mann–Whitney test). Right panel, Dissociation of the lipid-raft Orai1/TRPC1/SK3 channel complex by Ohmline decreased inhibits SOCE-dependent cell migration. Histograms showing HCT-116 cell migration treated with Ohmline +/− siSTIM1. The normalized cell number corresponds to the ratio of total number of migrating cells in presence of drugs/total number of migrating cells in control experiments. Results are expressed as mean ± SEM. **p<0.01, sample significantly different from control (N=3, n=9, Kruskal-Wallis test). C. Left panel, Dissociation of the lipid-raft Orai1/TRPC1/SK3 channel complex by Ohmline inhibits P-Akt-dependent cell migration enhanced by EGF. Results are expressed as mean ± SEM. ***p<0.001, sample significantly different from control (N=2, n=6 Kruskal-Wallis test). Right panel, Immunoblots show level of P-Akt in HCT-116 cells treated with Ohmline +/− EGF.P-Akt levels (standardized based on total Akt) was determined by densitometry scanning to generate the values shown in the bar graph. Results are expressed as mean ± SEM. *p< 0.5 and **p<0.01, sample significantly different from control (N=3, Kruskal-Wallis test). D. Dissociation of the lipid-raft Orai1/TRPC1/SK3 channel complexby Ohmline decrease Rac1 and calpain enhanced by EGF treatment. Left panel, activated Rac1 (standardized based on total Rac1) was determined by densitometric scanning to generate values shown in the bar graph. Results are expressed as mean ± SEM. *p<0.05 or **p<0.01, sample significantly different from control (N=6, Kruskal-Wallis test). Right panel, calpain activity results are expressed as mean ± SEM. *p<0.05 significantly different from control (N=4, Kruskal-Wallis test). E. Effect of cetuximab and panitumumab used in combination with Ohmline on cancer cell migration. Left panel, Histograms showing cell migration with cetuximab or panitumumab +/− Ohmline for 24h. Results are expressed as mean ± SEM. **p<0.1 and ***p<0.001, sample significantly different from control (N=3, n=9, Kruskal-Wallis test). Right panel, Immunoblots represent expression of P-Akt in HCT-116 cells treated with cetuximab +/− siSK3, Ohmline or MK2206.P-Akt levels (standardized based on total Akt) was determined by densitometry scanning to generate the values shown in the bar graph. Results are expressed as mean ± SEM. ***p<0.001, sample significantly different from control (N=3, Kruskal-Wallis test). F. Expression of SK3 protein in colon tissues. Representative images of cancer cells detected by SK3 immunostaining in primary tumors obtained from human colon cancer. A semiquantitative scoring was used to quantify SK3 levels on the basis of the data provided by the Human Protein Atlas (www.proteinatlas.org). Using this method, each staining/expression was scored as 0 (not detected), 1 (low intensity), 2 (medium intensity) or 3 (high intensity).