Figure 1.
miR-34a restores sensitivity to erlotinib in non-small cell lung cancer cells.
(A) Dose-dependent effect of erlotinib in parental HCC827 cells. Cells were treated with erlotinib in a serial dilution for 3 days, and cellular proliferation was determined by AlarmaBlue. (B) HCC827 cells resistant to erlotinib (HCC827res) were developed by incubating cells with increasing erlotinib concentrations over the course of 10 weeks until cells grew normally at concentrations equal to IC90 in parental HCC827. (C, D) HCC827res and H1299 cells were reverse-transfected with 0.3 nM miR-34a or miR-NC, and incubated in media supplemented with erlotinib in a serial dilution. After 3 days, cellular proliferation was determined. IC50 values of erlotinib alone or in combination with miRNA are shown in the graphs.
Figure 2.
Synergistic effects between miR-34a and erlotinib in NSCLC cells.
(A) Combination index (CI) analysis. CI values were generated by linear regression (red) and non-linear regression methods (blue). Trendlines indicate CI values at any given effect (Fa, fraction affected, % inhibition), and symbols represent CI values derived from actual data points. CI = 1, additivity; CI>1, antagonism; CI<1, synergy. (B) Isobologram analysis. The diagonal, dotted line indicates additivity, and the red symbol shows dose requirements to achieve 50% and 80% (A549, H1299, H460) or 30% and 50% (H226) cancer cell inhibition, respectively. Data points below the line of additivity indicate synergy, data points above denote antagonism. (C) Curve shift analysis. Data derived from non-linear regression trendlines were normalized to IC50 values of the single agents (IC50 eq) and plotted in the same graph. Left and right shifts of the dose-response curves of the combination (red dotted line) relative to the dose-response curves of the single agents (grey, black) indicate synergy or antagonism, respectively. Actual experimental data points are shown (symbols).
Figure 3.
Multiple ratios of erlotinib and miR-34a cooperate synergistically in A549 cells.
(A) Summary table showing potency (Fa), CI and DRI values of erlotinib and miR-34a combined at various concentrations and ratios. The molar miR-34-erlotinib ratios 1∶533, 1∶1333, 1∶3333 (IC50:IC50 ratio), 1∶8333, and 1∶20833 are indicated in yellow, green, red (IC50:IC50 ratio), blue and maroon. (B) Combination index plot of various drug ratios. CI values from actual data points are indicated by symbols. (C) Isobologram at 80% cancer cell inhibition. Colored symbols represent the 80% isobole of various ratios. The black line represents the isobole derived from actual erlotinib-miR-34a combinations that produced 80% (±2%) inhibition. (D) Curve shift analysis of various drug ratios.
Figure 4.
Erlotinib and miR-34a synergize in HCC cells.
(A) Combination index analysis. (B) Isobologram analysis. (C) Curve shift analysis. See legend to Figure 2 for explanation of graphs.