Antitumor activities of Quercetin and Green Tea in xenografts of human leukemia HL60 cells

Abstract

Quercetin is one of the most abundant flavonoids, present in fruits and vegetables and has been shown to have multiple properties capable of reducing cell growth in cancer cells. Green tea is a widely consumed beverage, known for a potential source of free radical scavenging and anti-cancer activities. Herein, we investigate the in vivo antitumor efficacy of quercetin and green tea in human leukemia. Human tumors were xenografted into NOD/SCID mice. Quercetin and green tea reduced tumor growth in HL-60 xenografts accompanied by decreased expression of anti-apoptotic proteins, BCL-2, BCL-XL and MCL-1 and increased expression of BAX, a pro-apoptotic protein. Moreover, caspase-3 was activated to a greater extent after quercetin and green tea treatment. Quercetin and green tea also mediated G1 phase cell cycle arrest in HL-60 xenografts. Treatment with quercetin and green tea induced conversion of LC3-I to LC3-II as well as activation of autophagy proteins, suggesting that quercetin and green tea initiate the autophagic progression. We have provided evidence that quercetin and green tea induces signaling at the level of apoptosis, cell cycle and autophagy which converge to antigrowth effects in HL-60 xenograft mice suggesting that these compounds may be a compelling ally in cancer treatment.

Figure 1

Quercetin (A) and (B) Green tea reduce tumor growth in HL60 xenografts. (C) Quercetin (D) and Green tea tumor volume after 21 days of treatment. The body weights of the xenograft mice treated with Quercetin (E) and Green tea (F). NOD/SCID mice with HL-60 tumors were treated or not with quercetin (120 mg/kg) once every 4 days or green tea (100 mg/kg) every day during 21 days. Every 7 days the tumor volumes were evaluated according to the following formula: tumor volume (mm³) ¼ (length × width2)/2. The control group received equal amounts of vehicle solution, as indicated in Materials and Methods. (Values are means ± SD (mean of 6 mice/group); *P < 0.05.

Figure 2

Quercetin and Green tea induces apoptosis of leukemic cells. NOD/SCID mice with HL60 tumors were treated or not with quercetin (120 mg/kg), once every 4 days or green tea (100 mg/kg), every day. After 3 weeks, tumors were harvested and relative levels of protein in tumor lysates were determined by Western blotting (A,B) BCL-2; (C,D) BCL-XL; (E,F) BAX; (G) -MCL-1; (H) -cytocrome c; (I) -p-JNK. (J,K) IHC analysis of cleaved caspase-3 in tumor sections and the percentage of caspase 3 positive nuclei of cells per field; four fields per tumor section. Counts were made using x40 objective connected to a light microscope (Olympus CBA; Olympus America). Values are means ± SD (mean of 6 mice/group); *P < 0.05.

Figure 3

Quercetin and Green Tea induces arrest of the G1 phase of cell cycle in HL-60 xenografts. NOD/SCID mice with HL60 tumors received or not quercetin (120 mg/kg), once every 4 days or green tea (100 mg/kg), every day. After 3 weeks, tumors were harvested and relative levels of protein in the tumor lysates were determined by Western blotting; (A,B) CDK6; (C,D) CDK4; (E,F) CDK2; (G,H) CYCLIN D; (I,J) CYCLIN E; (K,L) CYCLIN A; (M) p-RB. (N,O) IHC analysis of p21 protein in tumor sections and the percentage of p21-positive nuclei of cells per field; four fields per tumor section. Counts were performed using x40 objective connected to a light microscope (Olympus CBA; Olympus America). Values are means ± SD (mean of 6 mice/ group); *P < 0.05.

Figure 4

Quercetin and Green Tea induce autophagy in HL60 xenografts. NOD/SCID mice with HL60 tumors were treated or not with quercetin (120 mg/kg), once every 4 days or green tea (100 mg/kg), every day. After 3 weeks, tumors were harvested and relative levels of protein in tumor lysates were determined by Western blotting; (A,B) BECLIN-1; (C,D) PI3K; (E,F) ATG5-ATG12; (G,H) ATG7; (I,J) IHC analysis of cleaved LC3 in tumor sections and the percentage of LC3-positive nuclei of cells per field; four fields per tumor section. Counts were performed using x40 objective connected to a light microscope (Olympus CBA; Olympus America). Values are means ± SD (mean of 6 mice/group); *P < 0.05.