行政院國家科學委員會專題研究計畫成果報告
氧化砷治療癌症可行性之探討
Pr eclinical Study of Ar senic Tr ioxide in the Tr eatment of Solid Tumor
計畫編號：NSC 87-2314-B002-079
執行期限：86 年 8 月 1 日至 87 年 7 月 31 日
主持人：陳耀昌教授 楊志新醫師
執行機構及單位名稱台大醫院檢驗醫學部,台大醫院腫瘤醫學部
一、中文摘要 砷元素廣泛的散佈在自然界中。長期 的暴露會引起各種疾病，也會增加得到皮 膚癌、肺癌、膀胱癌等癌症的機會。但自 古以來，砷就被用來治療多種疾病，包括 關節炎、氣喘及白血病等癌症。 最近在哈爾濱及上海的血液科醫師分 別發現，注射氧化砷能治療對化學治療、 全反式維生素甲酸失效的急性前骨髓型白 血病病人。效果很好，毒性也可接受。 氧化砷可抑制細胞生長，它可干擾蛋白 質硫氫基(-SH)及磷酸化的功能，從而抑制 細胞代謝、結構。砷也可以破壞 DNA、改 變細胞的染色體。我們發現，氧化砷在病 人身上可達到的濃度下，即可抑制癌細胞 的生長，可能有臨床應用價值。 本計畫的目的在探討氧化砷用在各種 癌症治療的可行性，以及初步瞭解氧化砷 毒殺癌細胞及癌細胞抗藥性的可能機轉。 在本計畫中，我們發現氧化砷對膀胱癌 及白血病細胞有極高毒性，對卵巢癌及腸 胃癌症的細胞毒性為中等，對其他癌症之 效果則不佳。氧化砷對癌細胞之毒性似乎 是經由引起細胞凋亡之機轉。氧化砷應可 應用在固態腫瘤治療之臨床試驗。由於高 的 GSH 往往引起細胞的抗藥性，使用 BSO 可能可以將加強治療的效果。 關鍵字: 氧化砷，癌症治療，抗藥性 Abstr actArsenic is a ubiquitous element in environment. Chronic exposure of arsenic compound may cause various diseases and is also associated with increased risk of skin cancer, lung cancer, bladder cancer. Despite
these chronic toxicity, arsenic is an ancient medication and was used widely in both western and Chinese medicine. It has been used to treat asthma, arthritis and several types of cancer, including leukemias.
Arsenic trioxide has revived after the revelation that it is very effective in the treatment of acute promyelocytic leukemia patients who were refractory to both chemotherapy and all-trans retinoic acid.
Our preliminary cytotoxicity results indicated that at concentrations attainable in patient plasma, As2O3may inhibit the growth
of some solid tumor cell lines.
The objective of this project is to test feasibility of using As2O3 in the treatment of
solid tumors. The secondary aim is to find out mechanisms of toxicity and resistance of tumor cells to arsenic compounds.
In this study, we found that As2O3 is very
cytotoxic to bladder cancer and APL cells. It is moderately active in ovarian and gastrointestinal cancer cells. Other types of cancer are resistant to As2O3. As2O3 seems to
kill tumor cells through induction of apoptosis. Our results indicate that it is warranted to use As2O3 in clinical trials to
treat solid cancer patients, especially for bladder cancer. High GSH content was associated with intrinsic arsenic resistance in cancer cells. BSO may be considered to enhance cytotoxicity of arsenic treatment.
二、緣由與目的
Ar senic in envir onment
mineral, soils, air, plants and animals. Human exposed to arsenic from inhaled air, drinking water and seafood. Artesian wells in Tainan county contain up to 1.8mg/L of arsenic in contrast to less than 0.01mg/L in most of water supply1.
Ar senic in tr aditional medicine
Arsenic compounds have been used for a long time in both western and Chinese medicine. Fowler’s Solution contains As2O3
and was administered for the treatment of leukemia, psoriasis and asthma. Pearson’s solution (0.5% arsenate) can be used up to 20mg (4.8mg of As) per day. Salvarsan and neoarphenamine are arsenic compounds used for the treatment of syphilis.1 As2O3 is
present in 砒霜. 雄黃 contains As2S3. Daily
intake of arsenic may approach 10mg in some formulas.
Ar senic in the tr eatment of cancer
Local application of arsenic was used in the past in the treatment of cancer of skin, oral cavity, cervix etc. Doctors have been using arsenic in the treatment of chronic myelogenous leukemia, lymphoma, stomach cancer and esophageal cancer in Mainland China. However, there were no reports on its usefulness and efficacy in the treatment of cancer in the era of contemporary medicine.
As2O3 in the tr eatment of acute
pr omyelocytic leukemia (APL)
As2O3 has revived recently with the
revelation that arsenic may be the most effective treatment APL.
Chemotherapy and all-trans retinoic acid are two effective treatments for APL patients.
complete remission were observed in APL patients.3
Toxicity of As2O3 treatment include
infection 11.97%, nausea 22.41%, vomiting 8%, diarrhea 4.27%, numbness 9.4%, increase liver enzyme 4.27%, edema 1.71%. Chronic toxicities are few.
Mechanism of action of As2O3 in the
tr eatment of APL
As2O3 at concentrations achievable in
patients’ plasma (1-2µM) were found to kill NB4 cells, an APL cell line. There were no cytotoxic effect at the same As2O3
concentrations in other non-APL cell lines (HL60, U937). As2O3 induce apoptosis in
NB4 cells. Inhibition of oncoprotein PML-RARα was suggested. In addition, partial induction of differentiation was noted in NB4 cell treated with 0.25µM of As2O3 with
long period of incubation4.
Other mechanism of cytotoxicity of As2O3
Other mechanisms have been proposed to contribute to arsenic toxicity. Arsenic was known to induce DNA strand breaks associated with DNA-protein cross-links5, induce sister-chromatid exchanges6. Arsenic was also shown to inhibit DNA repair.7 It may interfere protein phosphorylation and inhibit the function of proteins carrying sulfhydryl group (-SH).
As2O3 plasma levels in human
Pharmacokinetics study was done in APL patients treated with daily infusion of 10mg As2O3. Peak levels were 4.2 to 6.7µM and
were reached at 3-4 hours after infusion. Drug concentrations in the blood were
glutathione-s-transferase pi (GST-π) over-expression were linked to arsenic resistance10. A mdr-1 homologue multidrug resistant-associated protein (MRP) transfectants of HeLa cells were shown to be resistant to arsenic11. A combination of overexpression of GST-π and MRP, therefore, may theoretically confer high resistance to arsenic in cancer cells12.
Specific aim
1. To study the feasibility of using As2O3 as
anticancer drug in solid tumors by cytotoxicity test.
2. To study the intrinsic resistance mechanism of As2O3 in cancer cells.
3. To study the mechanism of As2O3 toxicity
to cancer cells.
Impor tance of the r esult achieved:
As2O3 may be effective in the treatment of
solid tumor.
三、結果與討論
1. cytotoxicity of As2O3 in cancer cells.
Cancer cells were added to each well in 96-well plates for 24 hours. As2O3 was
diluted in water and added to each wells resulted in final concentrations from 30nM to 100µM. Cells were grown for 96 hours. MTT or SRB assays were performed to viable cells in each well after drug treatment. Cytotoxicity curves were shown below:
2. IC50s of As2O3 in these cells: Origin IC50s# BFTCC905 Bladder 0.34!0.03* NTU-B1 Bladder 0.49!0.16* NB4 APL 0.64!0.11* T24 Bladder 0.93!0.20* A2780 Ovary 1.12!0.33* SW620 Colon 1.16!0.15* AGS Stomach 1.16!0.20* TSCH8302 Cervix 2.50!0.69* MCF7 Breast 2.67!0.66* BFTCC909 Bladder 2.84!0.79* H460 Lung 3.27!0.49* A172 Glioblastoma 3.40!0.40* CL-1 Lung 4.17!0.50* Hep3B Liver 5.17!1.02* HepG2 Liver 7.17!1.20*
# in µM *average and standard error of at least 3 independent experiments
Thr ee gr oups of cancer can be identified. Gr oup1: bladder cancer , APL ar e ver y sensitive to As2O3.
Gr oup2: gastr ointestinal cancer ar e moder ately sensitive to As2O3.
Gr oup3: lung cancer , liver cancer , cer vical cancer , br east cancer and glioblastoma. Relatively r esistant to As2O3.
2. As2O3 induce apoptosis in NTU-B1 cells.
Cells were treated with 0, 0.3, 1 and 3µM of As2O3 for 48 and 72 hours. DNA were
isolated and subjected to gel electrophoresis. DNA ladder signifying apoptosis of the cells were observed in cells treated with 3µM for 2 days or 1µM for 3 days.
3. Glutathione content in cancer cells.
Glutathione content was measured by colorimetric assay. GSH content# NTUB1 7.59!1.17* H460 21.8!2.9* NB4 6.12!0.96* SW620 4.91!0.17* AGS 7.30!0.84* BFTC905 6.03!1.35* BFTC909 17.1!2.9* Hep3B 17.4!1.7*
#(µg/mg protein) *average and standard error of at least 3 independent experiments
There is good correlation between GSH content and cytotoxicity of As2O3 in cancer cells. Lower GSH content is associated with higher toxicity.
4. BSO deplete glutathione in cancer cells GSH content# NTUB1 7.59!1.17* NTUB1+10µM BSO 2.28!0.64* NTUB1+50µM BSO 1.00!0.44* H460 21.8!2.9* H460+10µM BSO 4.95!0.17* H460+50µM BSO 3.17!0.27*
#(µg/mg protein) *average and standard error of at least 3 independent experiments
5. BSO sensitize cancer cells to ar senic tr ioxide. IC50 of As2O3 in NTU-B1 cells
was 0.6 µM. IC50 was 0.2 µM when cells are co-incubated with 50 µM of BSO.
四、計劃成果自評
other hand, there were no correlation between glutathione transferase or MRP content and cytotoxicity. This finding lead to the strategy to use BSO to enhance arsenic toxicity to cancer cells or to reverse arsenic resistance.
五、參考文獻
1 Kuo T: Arsenic content of artesian well water in
endemic area of chronic arsenic poisoning. Rep. Inst. Pathol. Natl Taiwan Univ., 20:7,1968
2 孫鴻德﹐馬玲﹐胡喨晨﹐張亭棟: 癌零一號結合 中醫辨證治療急性早幼粒細胞白血病 32 例。中 國中西醫結合雜誌 12:170,1992 3 張鵬 “713”治療急性早幼粒細胞白血病 117 例﹐ 臨 床 觀 察 及 機 制 探 討 。 Chinese Journal of Hematology, 17(2):58,1996
4 Chen GQ, Zhu J, Shi XG et al. :In vitro studies on
cellular and molecular mechanisms of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia: As2O3 induces NB4 cell apoptosis with downregulation of Bcl-2 expression and modulation of PML-RARα/PML porteins. Blood 88:1052,1996
5 Dong JT, Luo XM: Arsenic-induced DNA-strand
breaks associated with DNA-protein crosslinks in human fetal lung fibroblasts. Mutation Research 302:97, 1993
6 Jha AN, Noditi M, Nilsson R, Natarajan AT:
Genotoxic effects of sodium arsenite on human cells. Mutation Research 284:215, 1992
7 Lee-Chen SF, Yu CT, Jan KY: Effect of arsenite on
the DNA repair of UV-irradiated Chinese hamster ovary cells. Mutagenesis 7:51, 1992
8
Shen ZX. Cehn GQ, Li XS et al. Use of arsenic trioxide in the treatment of APL: II. Remission indution in relapsed patients and pharmacokinetics. Blood 88,10(suppl 1):292a,1996
9 Vicker PP, Dixon RB, Cowan KH: A pleiotropic
response associated with resistance of breast cancer cells to antineoplastic drugs and hormonal agents.
