Anti-tumour activity and toxicological studies of combination treatment of Orthosiphon stamineus and gemcitabine on pancreatic xenograft model

Figure 1 Tissue sections of mice spleen stained with haematoxylin and eosin. Tissue sections from untreated and treated groups showed normal spleen architecture. A: Untreated; B: Gemcitabine (10 mg/kg per 3 d); C: C5EOSEW5050ESA (200 mg/kg per day); D: C5EOSEW5050ESA (200 mg/kg per day) and gemcitabine (10 mg/kg per 3 d) combination; E: C5EOSEW5050ESA (400 mg/kg per day); F: C5EOSEW5050ESA (400 mg/kg per day) and gemcitabine (10 mg/kg per 3 d) combination.

Figure 2 Tissue sections of mice kidneys stained with haematoxylin and eosin. Tissue sections showed normal glomeruli and tubules in all treated groups and untreated. A: Untreated; B: Gemcitabine (10 mg/kg per 3 d); C: C5EOSEW5050ESA (200 mg/kg per day); D: C5EOSEW5050ESA (200 mg/kg per day) and gemcitabine (10 mg/kg per 3 d) combination; E: C5EOSEW5050ESA (400 mg/kg per day); F: C5EOSEW5050ESA (400 mg/kg per day) and gemcitabine (10 mg/kg per 3 d) combination.

Figure 3 Tissue sections of mice liver stained with haematoxylin and eosin. Liver sections showed normal architecture with distinct hepatic cells, sinusoidal spaces, and a central vein in all treatment groups and untreated; mild hepatocellular swelling is seen in all groups except the C5EOSEW5050ESA (200 mg/kg per day) group where no swelling is present. A: Untreated; B: Gemcitabine (10 mg/kg per 3 d); C: C5EOSEW5050ESA (200 mg/kg per day); D: C5EOSEW5050ESA (200 mg/kg per day) and gemcitabine (10 mg/kg per 3 d) combination; E: C5EOSEW5050ESA (400 mg/kg per day); F: C5EOSEW5050ESA (400 mg/kg per day) and gemcitabine (10 mg/kg per 3 d) combination.

Figure 4 Tumour growth curve of Panc-1 cells in subcutaneous pancreatic tumour growth in nude mice and tumour weight. Treatments were given when the tumours reached 100 mm³. A: Untreated; B: Gemcitabine (10 mg/kg per 3 d); C: C5EOSEW5050ESA (200 mg/kg per day); D: C5EOSEW5050ESA (200 mg/kg per day) and gemcitabine (10 mg/kg per 3 d) combination; E: C5EOSEW5050ESA (400 mg/kg per day); F: C5EOSEW5050ESA (400 mg/kg per day) and gemcitabine (10 mg/kg per 3 d) combination. Combination treatment of 400 mg/kg C5EOSEW5050ESA and gemcitabine (F) synergistically reduced the tumour growth compared to a single treatment. The combination treatment of 400 mg/kg C5EOSEW5050ESA and gemcitabine (F) significantly reduced tumour weight compared to a single treatment. Error bars represent standard deviations. Statistics analysis (^aP < 0.05; ^bP < 0.01; ^cP < 0.001, two-way ANOVA for tumour growth, one-way ANOVA with Tukey’s HSD posthoc test for tumour weight, n = 6 animals per group) using SPSS and GraphPad Prism 6.0 software.

Figure 5 Tumour necrosis percentage in tumour tissues stained with haematoxylin and eosin. A: Untreated group; B: Gemcitabine (10 mg/kg per 3 d); C: Orthosiphon stamineus (O.s) (200 mg/kg per day); D: O.s (200 mg/kg per day) + gemcitabine (10 mg/kg per 3 d); E: O.s (400 mg/kg per day); F: O.s (400 mg/kg per day) + gemcitabine (10 mg/kg per 3 d); G: Estimated marginal means of necrosis. Combination treatment of 400 mg/kg C5EOSEW5050ESA with gemcitabine synergistically inhibited tumour necrosis compared to a single treatment. Arrows indicate the necrotic area in tumour sections. Error bars represent standard deviations. Statistics analysis (two-way ANOVA, n = 6 animals per group) using SPSS software. O.s: Orthosiphon stamineus.

Figure 6 Ki67 proliferation protein expression in treated and untreated groups. A: Untreated group; B: Orthosiphon stamineus (O.s) (200 mg/kg per day); C: O.s (400 mg/kg per day); D: Gemcitabine (10 mg/kg per 3 d); E: O.s (200 mg/kg per day) + gemcitabine (10 mg/kg per 3 d); F: O.s (400 mg/kg per day) + gemcitabine (10 mg/kg per 3 d); G: Estimated marginal means of Ki67. Single treatment either by C5EOSEW5050ESA or gemcitabine significantly reduced the Ki67 protein expression in tumour tissues compared to the untreated group. No significant reduction of Ki67 protein expression was noted between the groups treated by C5EOSEW5050ESA 200 mg/kg per day and the gemcitabine combination. Combination treatment of C5EOSEW5050ESA 400 mg/kg per day and gemcitabine synergistically reduced Ki67 protein expression in tumour tissue compared to a single treatment. Error bars represent standard deviations. Statistics analysis (Two-way ANOVA, n = 6 animals per group) using SPSS software. O.s: Orthosiphon stamineus.