The combined effect of Chinese medicinal extract polysaccharide peptide (PSP) and the chemotherapeutic agents-cytarabine, doxorubicin and etoposide in human leukemic cells and normal human T-lymphocytes

by Hui, Pui-yan

(Uncorrected OCR) Abstract of thesis entitled

"THE COMBINED EFFECT OF CHINESE MEDICINAL EXTRACT POLYSACCHARIDE PEPTIDE (PSP) AND THE CHEMOTHERAPEUTIC AGENTS-CYTARABINE, DOXORUBICIN AND ETOPOSIDE IN HUMAN LEUKEMIC CELLS AND NORMAL HUMAN T-LYMPHOCYTES"

Submitted by

PuiYanHUI

for the degree of Master of Philosophy at The University of Hong Kong

in August 2003

Polysaccharide-peptide (PSP) extracted from a Chinese medicinal

mushroom-Coriolus versicolor or Yunzhi has been reported to be beneficial to

cancer patients in combination with radiotherapy or chemotherapy. However, the

mechanistic interaction between PSP and these cancer treatments has not been fully

understood. In order to investigate the combined effect and interaction of PSP with

several anticancer drugs--cytarabine (Ara-C), doxorubicin (Doxo) and etoposide

(VP-16), in vitro experiments were carried out using HL-60 cells and normal human

T-Iymphocytes. Flow cytometry, ELISA, western blotting and microscopic

examination were applied in this study.

Cell cycle analysis by flow cytometry revealed that PSP, at low doses,

accumulated HL-60 cells at the S phase. Consequently, it is hypothesized that

subsequent exposure ofPSP-treated cells to S-phase specific chemotherapeutic agents

would enhance the cytotoxicity of these drugs. Annexin V binding assay consolidated

the observation that the combination ofDoxo or VP-16 with PSP exhibited synergism

in cytotoxicity on HL-60 cells whereas an antagonistic effect on the cell-killing action

of Ara-C was observed. Quantification of pre-G1 cells by flow cytometry demonstrated the dose effect of PSP on the cytotoxicity enhancement of Doxo and VP-16 on HL-60 cells. However, it was noticed that PSP did not interfere with the cell cycle nor inhibit the proliferation of normal human T -lymphocytes evaluated by flow cytometric DNA content analysis and BrdU proliferation assay, respectively. Annexin V binding assay provided further evidence that PSP did not sensitize normal human T-lymphocytes to either Doxo or VP-16.

By evaluating the cell cycle distribution, it was shown that the combined treatment of PSP with VP-16 or Doxo caused the elimination of mainly S-phase cells via apoptosis induction whereas the ability of Ara-C to remove S-phase cells was restrained by pretreatment with PSP. Possible factors contributing to the antagonism with Ara-C may be the removal of cells from the Gl-S boundary and/or the slowed down progression of HL-60 cells in the S phase by PSP treatment. Further investigation of the mechanistic interaction between PSP and VP-16 or Doxo revealed that up-regulation of topoisomerase IIa and cyclin E expression by PSP might be the possible mechanisms contributing to the induction of S-phase accumulation and sensitization ofHL-60 cells to Doxo and VP-16. In addition, flow cytometric analysis and western blotting demonstrated the implication of caspase 3 during apoptosis induced by combined treatment ofDoxo or VP-16 with PSP.

Summarizing the results ofHL-60 cells and normal human T-lymphocytes, it is concluded that PSP acts selectively on leukemic cells and the capability to promote the cytotoxicity of Doxo and VP-16 demonstrates the potential of PSP as a candidate in combination treatment with VP-16 and Doxo in leukemia.