Figure 1.
IC50 of Gemcitabine, Thymoquinone and Betulinic acid at 48 hrs.
IC50 value was calculated onHuman pancreas adenocarcinomacell lines (A) MIA PaCa-2 (B) PANC-1 and (C) Normal cell line FR2: For individual treatment of Gemcitabine, Thymoquinone and Betulinic acid for 48 hrs (**, significant as compaired to control at p value<0.01, n = 3) in-between the three cell lines.
Figure 2.
Effect of Betulinic acid, Thymoquinone and Gemcitabine alone and (in combination) on growth of human pancreatic tumor cell lines.
(A) MIA PaCa-2 and (B) PANC-1 cells were exposed to graded concentrations of Betulinic acid, Thymoquinone or Gemcitabine either alone or in combination at a fixed dose ratio of Betulinic acid vs. Gemcitabine and Thymoquinone vs. Gemcitabine for 48 hrs.(Gemcitabinehaving exposure for 24 hrs only in case of combination), Gemcitabine (squares),Betulinic acid and Thymoquinone(circle) Gemcitabine+ betulinic acid and Gemcitabine +Thymoquinone (triangle).(Mono therapy (BA, GCB, TQ) Versus Combinations(CI(GCB:BA), CI(GCB:TQ))(**, significant difference at p value <0.01, n = 3).
Figure 3.
Isolobolograms of the combinations of Gemcitabine with Betulinic acid or Thymoquinone.
(A and B) Showing isolobolograms analysis of the combination of gemcitabine, Betulinic acid or Thymoquinone in, (A) MIA PaCa-2 cells with a fixed drug ratio 1∶0.01 of GCB:BA and GCB:TQ(B) PANC-1 cells with a fixed drug ratio 1∶0.2 of GCB:BA and GCB:TQ. The individual doses of gemcitabine, Betulinic acid and Thymoquinone, to achieve 90% (straight line) growth inhibition (Fa = 0.90), 75% (hyphenated line) growth inhibition (Fa = 0.75), and 50% (crosses) growth inhibition (Fa = 0.50) were plotted on the x- and y-axes. Combination index (CI) values calculated using Calcusyn software is represented by points above (indicating- antagonism between drugs) or below the lines (indicating- synergy). (X symbol) ED50, (plus sign) ED75 and (open dotted circle) ED90 (Monotherapy (BA, GCB, TQ) versus combinations (CI (GCB: BA), CI (GCB: TQ)) (***, significant difference at p value<0.001, n = 3).
Table 1.
Mean IC50 Value of drug (GCB) and dietary molecules (BA and TQ) on three cell lines (MIA PaCa-2; PANC-1; FR2) at 48 hrs.
Table 2.
Combination Index (CI) at ED50, ED75, ED90 values of Dietary with drug combination on two pancreas cancer cell lines.
Table 3.
CI values of Dietary molecules (BA and TQ) in combination with Drug (GCB).
Figure 4.
Effect on Mitochondrial membrane potential by Gemcitabine, Betulinic acid and Thymoquinone alone and in combinations.
(A) MIA PaCa-2: Betulinic acid alone and in combination CI (GCB:BA) with Gemcitabine induced loss of mitochondrial membrane potential (ψ) that is not observed in Thymoquinone and Gemcitabine neither alone nor in combination CI (GCB:TQ). Cells were stained with Rhodamine-123 (5 mg/mL) for 1 hr and analyzed in FL-1 channel of flow cytometer. Data in all figures are representative of one of three similar experiments. (B) Similar studies were carried out in PANC-1 cell line, Betulinic acid alone induces more loss of mitochondrial membrane than in combination with Gemcitabine CI(GCB:BA) also no loss in mitochondrial membrane was observed when treated with Thymoquinone and gemcitabine alone or in combination CI(GCB:TQ) for 48 hrs.(Control verses Monotherapy (BA, TQ, GCB) and Control verses Combinations (CI(GCB:BA), CI(GCB:TQ)),(***, significant difference at p value<0.001, n = 3).
Figure 5.
Flow cytometric analysis of apoptotic cell population.
(A) MIA PaCa-2 cells (1×105) were incubated with indicated concentrations of Betulinic acid, Thymoquinone and Gemcitabinealone and in combination CI(GCB:BA), and CI(GCB:TQ) for48 hrs and analyzed for annexinV-FITC/PI staining to determine apoptotic cell populations as described in Materials and Methods Section. (B) Similar studies were carried out with in PANC-1 cells after treatment with indicated doses of Betulinic acid, Thymoquinone and Gemcitabine alone and in combination CI(GCB:BA), and CI(GCB:TQ) for 48 hrs.(Control versus monotherapy (BA, TQ, GCB) and control versus combinations (CI(GCB:BA), and CI(GCB:TQ)), (***, significant difference at p value<0.001, n = 3).
Figure 6.
(A) MIA PaCa-2 and (B) PANC-1 synergetic doses at Fa 0.75 of Betulinic acid and Thymoquinone were used alone and in combination CI(GCB:BA) and CI(GCB:TQ) with Gemcitabine.From the blots it was observed that there was a decrease in expression of PKM2 when treated in combination doses. (C) PANC1 cell line doses at Fa 0.5 of betulinic acid, Thymoquinone and Gemcitabine were used alone and in combination, and from the blots we observed that there was a decrease in Pro-Caspase-3 followed by decrease in PARP and a slight decrease is observed in Thymoquinone treated in PKM2.
Figure 7.
Specific activity of Pyruvate Kinase (PK)-M2 in both of human pancreatic tumor cell lines.
(A) MIA PaCa-2 cells treated with Betulinic acid, Thymoquinone and Gemcitabine alone and in combination, CI(GCB:BA) and CI(GCB:TQ) at 48 hrs. A decrease in the activity in treated cells followed with moredecreasein combination(s), CI(GCB:BA) and CI(GCB:TQ), as compared with the control was observed;(B)In PANC-1 cell line vice versa was observed. Control versus monotherapy (BA, TQ, GCB) and Control versus combinations (CI(GCB:BA) & CI(GCB:TQ)), (***, significant difference at p value<0.001, n = 3)