2.  Chemotherapy is a common strategy used to kill cancer cells.  However toxicity of chemotherapy drugs to normal cells limits their application.

3.  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 (10–40 mM) in combination with Trichostatin A (TSA) cooperatively induces cell death in human leukemia HL-60 cells (Chen and Kang, 2005)

4.  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

5. 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 anti- oxidative and antiinflammatory Properties. quercetin

6.  Besides the synergistic or additive inhibiting effects, the combined treatment also reduces the toxicity of chemotherapy due to the lower dose of each compound.

7.  Does quercetin affect the anticancer effect of TSA on human lung cancer cells?

8. A549 cells (p53 +/+ ) or H1299 cells (p53 -/- ) 1. Cell growth 2. Apoptosis 3. p53, Bax, Apaf-1, Bcl-2, cytochrome c, acetyl-H3/H4 protein expression 4. caspase-9/3 activity In vitro study TSA (25 ng/mL) alone or combined with quercetin (2 or 5 μM)

10. A549 H1299 Quercetin has a stronger enhancing effect on TSA- induced cell-growth arrest and apoptosis in A549.

11. p53 GAPDH (A) (B) Quercetin increases p53 protein expression in A549 cells.

12. Bax Cytochrome C (cytosol) GAPDH Apaf-1 GAPDH (cytosol) Cytochrome C (mitochondria ) GAPDH (mitochondria) control TSA 5Q 5Q+TSA caspase-9 caspase-3 Quercetin enhances TSA-induced apoptosis through the mitrochondria pathway in A549 cells.

13. control control TSA 5Q 5Q+TSA nontargeted + p53 siRNA (A) (B) Bax GAPDH Apaf-1 caspase-9caspase-3

14. These results indicate that regulation of the expression of p53 protein plays an important role in enhancing TSA-induced apoptosis in A549 cells.

15. In Vivo study lls

16. (A) (B) IHC Staining for p53 controlLT LT+QHT control LT HT LT+Q p53 GAPDH Quercetin administrated by IP injection enhances anticancer effect of TSA in tumor- bearing mice.

17. TSA Quercetin p300 apoptosis associated gene apoptosis Quercetin enhances the anticancer effect of TSA in vitro and in vivo possibly through a p53-dependent pathway. H1299

18. Nude mice oral i.p. injection Quercetin TSA ？ 18 Enhancing effect Does quercetin given orally also has the same effect ?

19. 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. HPLC chromatograms in the plasma of nude mice.

20. acclimated for 1 week Week 2 ： subcutaneously injected A549 cells : 5x10 6 cells 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. Week 20 animals were sacrificed and analyzed 20 Nude mice In vivo study

22. Quercetin given orally at the doses, 20 and 100 mg/kg 3 times/week, fail to enhance the anticancer effect of TSA.

23. The contributing factor for the ineffectiveness of oral quercetin administration could be associated with the metabolic conversion of quercetin in vivo. Total or individual concentrations of quercetin and its metabolites in plasma (A) and (B) tumor tissues of tumor-bearing mice. Plasma Tumor tissues

24. Q3G significantly enhances the antiproliferation effect of TSA in A549 cells, however, the enhancing effect is less than that of quercetin. In vitro study

25. (A) (B) C OH+TSAIH+TSA TSA Quercetin given orally decreases the oxidative stress induced by TSA. The effect is similar to that of quercetin given by i.p. injection. Lymphocyte DNA Damage Lipid peroxidation

26. 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.

27. acclimated for 1 week Week 2 ： subcutaneously injected A549 cells : 5x10 6 cells 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. Week 19 animals were sacrificed and analyzed 27 Nude mice In vivo study These diet gave quercetin about 5-, 50-fold compared with the last study.

28. Similar to IQ+TSA, OH+TSA significantly decreases tumor growth in A549-tumor-bearing nude mice. TSA OH+TSA IQ+TSA (B) Tumor tissues p53 GAPDH C TSA OL OH IQ (A) TSA+

29. (A) (B) OL and OH decrease TSA-induced DNA damage and muscle weight loss.

30. Does quercetin enhance the anticancer effect of cisplatin, a widely used chemotherapy drug?

31. acclimated for 1 week Week 2 ： subcutaneously injected A549 cells : 5x10 6 cells 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. Week 20 animals were sacrificed and analyzed 31 Nude mice In vivo study

32. Cis2 in combination with HQ and IQ rather than Cis2 alone significantly inhibit tumor growth. The combined effects are similar to that of cis5. CIS2 CIS2+HQ CIS2+IQ CIS5

33. In A549 cells Both Quercetin and quercetin glucuronide significantly enhance the antiproliferation effect of cisplatin (1 μM). QuercetinQuercetin-3-glucuronide

34. CIS+Q increases the cells in sub-G1phase (apoptosis). CIS markedly induces cell cycle arrest in G2/M phase. Q3G only enhances the cell cycle arrest effect of CIS. CIS CIS+Q CIS+Q3G

35. 12 h C CIS 5Q 5G CIS +5Q CIS +5G P21 GAPDH P21 GAPDH 24 h P53 GAPDH P53 GAPDH 12 h 24 h 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.

36.  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.

37. Acknowledgement Dr. Shu-Ting Chan