Combination of anticancer drugs with quercetin in human lung ...

Combination of anticancer drugs with quercetin in human lung ...

Chemotherapy is a common strategy used to kill cancer cells. However toxicity of chemotherapy drugs to normal cells limits their application. Several phytochemicals, such as quercetin, have been reported to prevent cancer development by themselves or by enhancing the effects of anticancer drugs. For example, it has been shown that quercetin (1040 mM) in combination with Trichostatin A (TSA) cooperatively induces cell death in human leukemia HL-60 cells (Chen and Kang, 2005) TSA and vorinostat are histone deacetylase inhibitors (HDI), which are a promising class of anticancer drugs because they selectively induce the differentiation and apoptosis of various transformed cells. TSA Vorinostat Quercetin, a flavonoid, is a phytochemical found in various vegetal foods, such as

onions, apples, and green leafy vegetables. It has been suggested to possess antioxidative and antiinflammatory Properties. quercetin Besides the synergistic or additive inhibiting effects, the combined treatment also reduces the toxicity of chemotherapy due to the lower dose of each compound. Does quercetin affect the anticancer effect of TSA on human lung cancer cells? In vitro study A549 cells (p53+/+) or H1299 cells (p53-/-) TSA (25 ng/mL) alone or combined with quercetin (2 or 5 M) 1. Cell growth 2. Apoptosis 3. p53, Bax, Apaf-1, Bcl-2, cytochrome c, acetyl-H3/H4 protein expression 4. caspase-9/3 activity Quercetin has a stronger enhancing effect on TSAinduced cell-growth arrest and apoptosis in A549. A549 180 160 c 140

160 100 c c b ab 80 a b 60 b cell growth (%) cell growth (%) c 120 20 0 TSA ab a ab a a a

a 60 a 40 5Q a 80 20 2Q a a 100 40 control 24 h 48 h b 140 120 0 b

180 24 h 48 h c c H1299 200 2Q+TSA 5Q+TSA control 2Q 5Q TSA 2Q+TSA 5Q+TSA 35 35 A549 24 h 48 h 30 # c

20 b 15 c 10 a a b a a Apoptosis cells (%) Apoptosis cells (%) 24 h 48 h H1299 25 25 5 30 20 15 10 5 a

a a a a a a # b 0 0 control TSA 5Q 5Q+TSA control TSA 5Q 5Q+TSA (A) p53 GAPDH

p 5 3 p r o t e in e x p r e s s io n ( r e la t iv e in te n s ity ) Quercetin increases p53 protein expression in A549 cells. 200 # c b 150 100 a a control TSA 50 0 5Q 5Q+TSA 35 (B) - p53 siRNA transfection + p53 siRNA transfection Apoptosis cells (%)

30 25 20 15 c* b 10 a a 5 0 C TSA 5Q 5Q+TSA Quercetin enhances TSA-induced apoptosis through the mitrochondria pathway in A549 cells. control TSA 5Q 5Q+TSA Bax Apaf-1 GAPDH control

TSA 5Q 5Q+TSA Cytochrome C (cytosol) GAPDH (cytosol) Cytochrome C (mitochondria) GAPDH (mitochondria) caspase-3 caspase-9 b 200 ab 150 100 a a 50 0 b 300 250 control

TSA 5Q 5Q+TSA Relative caspase-3 activity (%) Relative caspase-9 activity (%) 300 200 a 100 0 a control a TSA 5Q 5Q+TSA (A) + p53 siRNA nontargeted control control 5Q+TSA

TSA 5Q Bax Apaf-1 GAPDH (B) caspase-9 caspase-3 350 250 - p53 siRNA transfection + p53 siRNA transfection 300 - p53 siRNA transfection + p53 siRNA transfection 150 * * 100 50 Relative caspsae-3 activity Relative caspsae-9 activity 200

250 200 150 * 100 50 0 control TSA 5Q+TSA 0 control TSA 5Q+TSA These results indicate that regulation of the expression of p53 protein plays an important role in enhancing TSA-induced apoptosis in A549 cells. In Vivo study lls Quercetin administrated by IP injection enhances anticancer effect of TSA in tumorbearing mice. 3500 control

LT HT LT+Q T um or volum e (m3)m 3000 (A) 2500 b b 2000 1500 ab 1000 a 500 0 0 1 2 3 4 5 6

7 8 9 10 11 12 13 14 15 16 17 18 weeks control (B) IHC Staining for p53 HT HT LT+Q p53 GAPDH LT LT+Q p 5 3 p r o te in e x p r e s s io n (% ) control LT 400 d c 300 b 200

100 a 0 control LT HT LT+Q Quercetin enhances the anticancer effect of TSA in vitro and in vivo possibly through a p53-dependent pathway. H1299 TSA Quercetin apoptosis associated gene p300 apoptosis Does quercetin given orally also has the same effect ? Nude mice Nude mice oral Quercetin Nude mice

TSA Nude mice Nude mice Nude mice Enhancing effect i.p. injection 18 It is known that after quercetin intake, conjugated metabolites, such as quercetin glucuronides and quercetin sulfates rather than quercetin aglycone are mainly present in human plasma. Q3G Q3'S Q methylquercetin OH+TSA baseline 8 10 12 14 16

18 20 22 24 26 Time (min) HPLC chromatograms in the plasma of nude mice. In vivo study Week 2 subcutaneously injected A549 cells : 5x106 cells acclimated for 1 week Week 20 animals were sacrificed and analyzed Week 5 Randomly assigned (6 groups) Control TSA : 0.5 mg/kg B.W., 2 times/week, i.p. OL : 20 mg/kg B.W., 3 times/week, gavage OH : 100 mg/kg B.W., 3 times/week, gavage IL : 2 mg/kg B.W., 3 times/week, i.p. IH : 10 mg/kg B.W., 3 times/week, i.p. Nude mice 20 Quercetin given orally at the doses, 20 and 100 mg/kg 3 times/week, fail to enhance the anticancer effect of TSA.

5000 C TSA OL+TSA OH+TSA IL+TSA IH+TSA Tumor size (mm3) 4000 3000 2000 * * * *# # #* * * * * * * # # # # # 1000 # # # 0 0 1 2 3

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 M)( Quercetin/quercetun metabolites concentration The contributing factor for the ineffectiveness of oral quercetin administration could be associated with the metabolic conversion of quercetin in vivo. 10 baseline OH+TSA IH+TSA * 8 * 4 a 2

* * Q3'S Q 0 Q3G 25 30 c b 25 b 20 a a a 10 5 0 C TSA OL+TSA OH+TSA IL+TSA IH+TSA

Quercetin/quercetin metabolites concentration (n mole/g tissue) Total quercetin concentration (n mole/g tissue) 35 15 Plasma 6 20 15 C TSA OH+TSA IH+TSA * Tumor tissues * 10 * 5 * * 0 Q3G

Q3'S Q Total or individual concentrations of quercetin and its metabolites in plasma (A) and (B) tumor tissues of tumor-bearing mice. In vitro study Q3G significantly enhances the antiproliferation effect of TSA in A549 cells, however, the enhancing effect is less than that of quercetin. 140 120 d d cell growth (%) 100 d 80 c 60 b 40 a 20 0 C

Q Q3G TSA Q+TSA Q3G+TSA Quercetin given orally decreases the oxidative stress induced by TSA. The effect is similar to that of quercetin given by i.p. injection. (A) Lymphocyte DNA Damage C Lipid peroxidation (B) TSA OH+TSA TBARs (Malondialdehyde nmole/ml) 0.7 IH+TSA 60 b DNA damage (% DNA in tail) 50 40 d

0.6 c 0.5 bc ab 0.4 a 0.3 0.2 0.1 0.0 C 30 20 10 bc a a a a a 0 C

TSA OL+TSA OH+TSA IL+TSA IH+TSA TSA OL+TSA OH+TSA IL+TSA IH+TSA Summary The study showed that quercetin given by gavage (20 and100 mg/kg body weight, 3 times/week; about 0.0015 and 0.0075 g/week) does not enhance the antitumor effect of TSA in tumor bearing mice. We postulate that the oral quercetin doses used are too low to exert the enhancing effect, because quercetin-3-glucuronide still has some enhancing effect on TSA-induced apoptosis. In vivo study Week 2 subcutaneously injected A549 cells : 5x106 cells acclimated for 1 week Nude mice Week 19 animals were sacrificed and analyzed Week 4 Randomly assigned (5 groups) Control TSA : 0.5 mg/kg B.W., 2 times/week, i.p. OL : 0.1% quercetin diet OH : 1% quercetin diet IQ : 10 mg/kg B.W., 3 times/week, i.p. These diet gave quercetin

about 5-, 50-fold compared with the last study. 27 Similar to IQ+TSA, OH+TSA significantly decreases tumor growth in A549-tumor-bearing nude mice. (A) (B) Tumor tissues TSA+ C 4000 C TSA OL+TSA OH+TSA IQ+TSA OL OH IQ p53 TSA GAPDH 2000 400 *# *# #* *# *

* #* #* # 1000 * #* # * # 0 0 2 4 6 8 10 12 OH+TSA IQ+TSA * * * * * * * # # # # # # # *# * * #* *# *# # #* # 14 16

18 20 22 p53 protein expression (relative intensity) Tumor size (mm 3) 3000 TSA *# 300 *# 200 100 0 C TSA OL+TSA OH+TSA IQ+TSA (A) (B)

50 40 # 30 20 * 10 * * 0 C TSA OL+TSA OH+TSA IQ+TSA Muscle weight/body weight (%) DNA damage (% DNA in tail) 140 * 120 * *

100 # 80 60 40 20 0 C TSA OL+TSA OH+TSA IQ+TSA OL and OH decrease TSA-induced DNA damage and muscle weight loss. Does quercetin enhance the anticancer effect of cisplatin, a widely used chemotherapy drug? In vivo study Week 2 subcutaneously injected A549 cells : 5x106 cells acclimated for 1 week Week 20 animals were sacrificed and analyzed Week 5 Randomly assigned (6 groups) Control cisplatin : 2 or 5 mg/kg, 1 time/week, i.p. OL : 0.1% quercetin diet OH : 1% quercetin diet

IQ : 10 mg/kg B.W., 3 times/week, i.p. Nude mice 31 Cis2 in combination with HQ and IQ rather than Cis2 alone significantly inhibit tumor growth. The combined effects are similar to that of cis5. 1600 control CIS2 CIS5 CIS2+LQ CIS2+HQ CIS2+IQ tumor size (mm 3) 1400 1200 1000 b b b a 800 400 200 * 0 1

2 3 4 5 6 7 * * * * * * * * * * * * * * 600 0 CIS2 8 * a a

9 10 11 12 13 14 15 16 17 weeks CIS2+HQ CIS2+IQ CIS5 In A549 cells Both Quercetin and quercetin glucuronide significantly enhance the antiproliferation effect of cisplatin (1 M). Quercetin g h cell growth (%) 100 140 f fg de 24hr 48hr e d d b b a 60

Quercetin-3-glucuronide h 40 24hr 48hr g g de 100 c 80 e f de cd 80 bc ab ab a 60 40 20 20 0

120 cell growth (%) 120 0 control CIS 2Q 5Q CIS+2Q CIS+5Q control CIS 2G 5G CIS+2G CIS+5G CIS markedly induces cell cycle arrest in G2/M phase. CIS+Q increases the cells in sub-G1phase (apoptosis). Q3G only enhances the cell cycle arrest effect of CIS. CIS

CIS+Q CIS+Q3G C 12 h 24 h CIS 5Q 5G CIS +5Q CIS +5G P21 GAPDH P21 GAPDH 12 h P53 GAPDH 24 h P53 GAPDH Cisplatin in combination with quercetin or quercetin glucuronide increases the p21 and p53 protein expression. Consistent with our previous results, the efficiency of quercetin is better than that of quercetin glucuronide. Conclusion Quercetin given orally or by i.p. injection may enhance the anticancer effect of TSA or cisplatin in vivo.

The enhancing effects of quercetin given orally are less than that by i.p. injection, because of the metabolic conversion. The mechanisms by which quercetin exerts its effect are associated with upregulation of p21 and p53 expression in human lung cancer cells. Acknowledgement Dr. Shu-Ting Chan

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