電化學療法治療鱗狀細胞癌及其細胞死亡機轉之探討

行政院國家科學委員會補助專題研究計畫成果報告

※※※※※※※※※※※※※※※※※※※※※※※※※

以電化學療法提高生體外抗癌藥物之細胞毒性

※※※※※※※※※※※※※※※※※※※※※※※※※

計畫類別：□個別型計畫 □整合型計畫

計畫編號：NSC89－2314－B－006－189－

執行期間：89 年 8 月 1 日至 90 年 7 月 31 日

計畫主持人

：

共同主持人：謝達斌、李玉雲、沈茂昌、許漢銘

本成果報告包括以下應繳交之附件：

□赴國外出差或研習心得報告一份

□赴大陸地區出差或研習心得報告一份

□出席國際學術會議心得報告及發表之論文各一份

□國際合作研究計畫國外研究報告書一份

執行單位：

中 華 民 國 90 年 10 月 29 日

行政院國家科學委員會專題研究計畫成果報告

以電化學療法提高生體外抗癌藥物之細胞毒性

Enhancement of cytotoxicity of anticancer agents mediated by electr ochemother apy in vitro

計畫編號：NSC 89-2314-B-006-189 執行期限：89 年 8 月 1 日至 90 年 7 月 31 日

主持人：王德華 執行機構及單位名稱：國立成功大學皮膚科 E-mail：twwong@mail.ncku.edu.tw

共同主持人：謝達斌¹、李玉雲²、沈茂昌³、許漢銘²

執行機構及單位名稱:國立成功大學牙科¹、皮膚科²、高雄醫學大學外

科³

一、中文摘要

電穿孔是經由給予細胞很短,但很 強的電脈衝造成細胞膜屏障暫時性減弱的 一種物理現象,在生體內及生體外都曾經被 用作促進螢光分子,DNA 及藥物進入細胞 的一種方法,若以此方法來促進化學治療藥 物進入細胞,則稱作電化學療法(簡稱 ECT), 電化學療法在臨床上曾用以治療惡性黑色 素瘤,基底細胞癌及頭頸部腫瘤, 得到相當 高的療效(objective response:85-99%;

CR:60%),但是臨床上用作 ECT 的藥物僅局 限於 Bleomycin 及 Cisplatin 兩種,尋找及開 發其他更有效,副作用低的化療藥物,仍然 是科學家努力的方向;Methotrexate 是葉酸 的衍生物,長久以來被用作治療各種癌症, 我們的共同主持人沈教授曾使用動脈內注 射 Methotrexate 治療超過 3000 例晚期頭頸 部腫瘤,到不錯的療效,因此,本研究探討 Methotrexate 與 Bleomycin 在不同癌細胞細 胞株中,電穿孔是否能提高化療藥物的細胞 毒性。

我們總共使用了 8 種細胞株,內含 5 株 鱗狀細胞癌,一株基底細胞癌,一株鼠類大 腸癌及一株鼠類黑色素瘤,其中兩株人類口 腔癌及一株基底細胞癌乃取自台灣本土病 人病變培養而成,結果顯示在所有測試的細 胞株中,Bleomycin 的 IC50在電穿孔後可以 降低 56 到 5012 倍,而以 Methotrexate 作為

ECT 的藥物測試中,則有 5 株細胞株的 IC50

可以降低 1.3 到 6.3 倍,在 Methotrexate 低濃 度(10^-7到 10^-10M)時,所有測試的細胞株,其 細胞毒性經由 ECT 可增強百分之十到三十, 這種在多種細胞株皆有反應的現象,說明了 Methotrexate 有可能在生體內也有類似效

果,由於低濃度 Methotrexate 是用作治療乾 癬及類風濕性疾病的有效藥物,也許以低濃 度 Methotrexate 的 ECT 可作為未來臨床上 之應用。

總括來說,我們的研究建立了電化學療 法藥物的一個簡單有效的篩檢模式,而更重 要的是我們測試了本土口腔癌及基底細胞 癌細胞株,結果顯示在生體外給予電化學療 法後,能大幅提高化療藥物的細胞毒性,為 未來臨床治療建立了重要的基礎。

關鍵詞：電穿孔、電化學療法、Bleomycin、

Methotrexate、鱗狀細胞癌、基底細胞癌。

Abstr act

Electroporation is a physical method that temporarily reduces cell membrane barriers by exposing cells to very brief but intense electric pulses. It has been used to facilitate the internalization of fluorescent molecules, DNA and drugs by cells in vitro and in vivo. The use of electric fields to mediate the uptake of chemotherapeutic agents, electrochemotherapy (ECT), has been used to treat tumors in clinical trials with very high objective (85-99%) and complete response rates (60%) for melanoma, basal cell carcinoma, and head and neck cancers.

The majority of clinical ECT studies have limited to the drug bleomycin or cisplatin.

Searching other chemotherapeutic agents that may potential candidate for ECT is still undergoing. Methotrexate, a folate analogue, has long been used as an anticancer agent to treat different kinds of cancer. Dr. Sheen, one of the co-investigators, has used intra-arterial

with advance head and neck cancer with high success rate. To study whether

electroporation can enhance the cytotoxicity of MTX and Bleomycin in different cancer cell lines, we compare the in vitro

cytotoxicity of these agents before and after electroporation.

Totally 8 cancer cell lines were tested. It included 5 squamous cell carcinoma cell lines, two of them were derived from

Taiwanese patients; one basal cell carcinoma cell line derived from our own patient, one murine colon carcinoma and one murine melanoma cell line. Results from all cell types indicate that the IC₅₀ of bleomycin can be reduced by a factor of 56 to 5012. The IC50 value of methotrexate could be reduced by factors from 1.3 to 6.3 in 5 cell lines as a result of electroporation. These IC50

reductions in multiple cell lines suggest that methotrexate may be effective in vivo as part of ECT treatment. In all eight tested cell lines, a 10-30% enhancement of cytotoxicity can be achieved when low concentration (10^-7 to 10^-10M) of methotrexate was combined with electric fields. The results imply that clinical application of methotrexate mediated ECT to treat psoriasis and other rheumatoid diseases is possible since lower MTX drug plasma level has been used clinically for benign diseases.

In summary, our study has established a simple and effective way to screen candidate chemotherapeutic agents for ECT. The promising results in treating cell lines

derived from our patients suggests ECT with bleomycin or methotrexate might become an alternative treatment in our oral cancer and basal cell carcinoma patients.

Keywor ds: Electroporation, electrochemotherapy, bleomycin,

methotrexate, squamous cell carcinoma, basal cell carcinoma

二、緣由與目的

Electrochemotherapy (ECT) is a new antitumor treatment based on the large potentiation of the antitumor effect of chemotherapeutic agents by the local

delivery of permeabilizing electric pulses (1).

electropermeabilization (also termed electroporation) of the cells in the tissue, allowing an increased drug delivery inside the tumors cells (2). Consequently the chemotherapeutic agents effects are greatly enhanced in the electric pulses-treated tumor, in the absence of side effects. Although ECT appears to be a promising technique for treating cancer, it is still an investigational approach. Among dozens of anticancer agents being tested, only bleomycin and cisplatin have entered into clinical trials (3).

Searching for new chemotherapeutic agent that is less cytotoxic to normal host cells, more cost-effective is still undergoing.

Methotrexate (MTX) is the most common used antimetabolite agent in cancer treatment. Professor Sheen of the Kaoshiung Medical College, one of the co-investigators, has treated more than 3000 patients with advance solid tumors with intra-arterial MTX chemotherapy with promising results (4). In this study, we compare the cytotoxicity of MTX mediated ECT with Bleomycin mediated ECT in different cell lines

including human squamous cell carcinoma, human basal cell carcinoma, murine colon carcinoma and murine melanoma.

三、結果與討論

Electroporation alone, especial with high electric field can lead to cell death. To assure that the cytotoxic effects of the anticancer drugs was truly due to the agent and not due to the electric treatment, the initial phase of this study focused on identifying electric conditions that induce electroporation for all 8 cell lines. A 90% of cell survival was determined for each cell line by exposing to six different fields ranging from 500 to 2500 V/cm. Three different samples were exposed to each field.

Twenty-four hours after exposure, an MTT (Thiazoyl Blue; Sigma USA) survival assay was performed. Survival data was pooled from these three replicated experiments for each cell lines to obtain a mean and SEM for each electric field. Figure 1 shows the resulting data for the human basal cell carcinoma cell line. A negligible effect on survival resulted when fields were less than

500 V/cm. However, survival was effected for fields of 900 V/cm and higher. The remaining cell lines had similar survival profiles at lower fields, but the threshold value at which survival began to decrease differed from cell line to cell line (Table 1).

The fields ranged from 550 to 1150 V/cm.

They are similar to other cancer cell lines in ECT study (3). These conditions were identified for further investigations as they were likely to result in a high degree of electroporation while maintaining viability.

Pulsed electric fields that provide 90%

of cell survival were used to facilitate drug entry into the cells. Each cell line was exposed to Bleomycin or MTX with and without electroporation. Eight drug

concentrations ranging from 1x10^-10 to 1x10^-3 M were tested. Cells (5x10⁶/ml) were

suspended in a B+K medium (5) with different concentrations of drugs. Three aliquots of cells in 150 ul were placed into separate electroporation cuvettes chamber (VWR Scientific Products, 58017-847 NY).

A high power pulse generator (Model 345, Velonex, Santa Clara, CA) was used to supply all rectangular high voltage direct current pulses. Amplitude and shape of each pulse delivered was recorded by a digital storage oscilloscope (Model 6121, Iwatsu Electronics, Tokyo, Japan). Electrically treated samples received eight 99 us pulses with a duty cycle of 1 pulse per second.

Electric field strengths were varied by adjusting the output voltage of the generator.

Two gold-plated electrodes are inserted within the cuvette chamber. The distance of the two parallel electrodes is 4 mm in our case.

The ratio of IC50 for samples that were not treated with electroporation (EP-) to the IC50 of samples treated with pulses (EP+) was used as a measure of cytotoxicity enhancement. Figure 2 shows results for Colon 26 cells exposing to MTX or

Bleomycin followed by electroporation. All cell lines show enhancements of cytotoxicity ranged from 56 to 5012 when Bleomycin was combined with electric pulses. This is

consistent with other studies (3). Six out of 8 cell lines show enhanced cytotoxicity to a lesser extent, after electroplated the cancer

cells with MTX (Table 1). It ranged from 1.3 to 6.3.

Cultured cells in a folic acid free medium enhanced 20-30 % cytotoxicity in MTX mediated ECT (Fig. 2). It agrees with previous studies that folic acid inhibited the influx of MTX (6). Although the cytotoxic effects of MTX were enhanced to a lesser extent when compared with Bleomycin, all cell lines show a 10-30 % enhanced effect at the lower MTX concentrations range from 10^-10 to 10^-7M. Figure 3 shows representative results for two combinations of cell line and drug. All other six cell lines tested in this study show the similar pattern when treating cells with low concentrations of MTX and electroporation. At a lower concentration, MTX enters cells mainly via a saturated carrier, and by diffusion at the higher

concentration (7). Electroporation may let the drug entering cells bypassing the carrier pathway. At a higher concentration, on the other hand, the amount of MTX that entered the cells by diffusion may equal to the quantity that entered as a result of drug exposure followed by electroporation.

Plasma level of less than 10^-8M is usually used for treating benign diseases such as psoriasis and rheumatoid diseases.

Electroporation after local administration of MTX has the benefit of providing high local concentration with minimal systemic side effects. Since electroporation can significant enhance cytotoxicity of MTX at low

concentrations as we shown here, MTX mediated ECT may has the potential for treating these diseases clinically.

MTX plasma level of 10^-5 to 10^-6 M has been used in treating different

malignancies (7). Kuriyama et al. showed that cisplatin mediated ECT can lead to complete regression in 50% of the mice with established Colon 26 tumor although the in vitro cytotoxicity was enhanced only 1.2 fold by electroporation (8). In our study,

electroporation can enhance the cytotoxicity of MTX to 6.3 fold in colon 26 cells compare to drug exposure alone. Further experiments are needed to clarify whether the in vivo MTX-ECT effect correlates with the in vitro results.

Basal cell carcinoma is one of the

around the world. The combination of

bleomycin with electric pulses can reduce the IC50 value by a factor of 5012 in the human basal cell carcinoma cells derived from our patient. Among the OECM-1, OC-2, Cal27 and SCC-15 human oral squamous cell carcinoma cell lines, OECM-1 and OC-2 are derived from Taiwanese patients. We have demonstrated that Bleomycin mediated ECT can enhance the cytotoxicity of Bleomycin to 85 and 314 fold in these cells. Oral cancer has become the second highest among all malignancies and the incidence is rising in Taiwan. The average 5-year survival rate is only 35%~50% under traditional treatment.

Our study is the first report to test oral cancer cell line derived from our patients with ECT.

The positive in vitro responses in the oral cancer cells and basal cell carcinoma cells shed light on further in vivo studies and suggest that this treatment might become a useful approach for treating these patients.

四、計畫成果自評

We have established a simple, effective in vitro model for the screening of candidate drugs for electrochemotherapy. Results from the basal cell carcinoma cells derived from our patient indicate that the IC50 of

bleomycin can be reduced by a factor of 5012 when electroporation is used to

facilitate internalization. In oral cancer cells derived from our patients, the IC₅₀ of bleomycin can be reduced by a factor of 85 and 314. The results suggest that

electrochemotherapy with bleomycin may be a promising alternative treatment for our basal cell carcinoma and oral cancer patients.

On the other hand, the IC₅₀ value of methotrexate could be reduced by factors from 1.3 to 6.3 in five cell lines as a result of electroporation. These IC₅₀ reductions in multiple cell lines suggest that methotrexate may be effective in vivo as part of ECT treatment. A 10-30% enhancement of cytotoxicity can be achieved when low concentration (10^-7 to 10^-10M) of

methotrexate was combined with electric fields in all eight tested cell lines. The results imply a possibility for clinical application of methotrexate ECT in treating psoriasis and

electrochemotherapy experiments in animal models can validate our in vitro results.

五、參考文獻

1. Mir LM, Orlowski S, Belehradck J Jr, et al. Biomedical application of electric pulses with special emphasis on antitumor electrochemotherapy.

Bioelectrochem Bioenerg 1995;

38:203-7.

2. Heller R, Jaroszeski M, Leo-Messina J, et al. Treatment of B16 mouse melanoma with the combination of

electropermeabilization and

chemotherapy. Bioelectrochem Bioenerg 1995;26:83-7.

3. Jaroszeski MJ, Dang V, Pottinger C, et al.

Toxicity of anticancer agents mediated by electroporation in vitro. Anti-cancer Drugs 2000; 11:201-8.

4. Sheen MC. Arterial infusion

chemotherapy in far advanced cancer. Jpn.

J Cancer Chemother 1994; 21:2089-100.

5. Hui Sw, Stoicheva N, Zhao YL.

High-efficiency loading, transfection, and fusion of cells by electroporation in two-phase polymer system. Biophys J 1996; 71:1123-30.

6. Sirotnak FM, Donsbach. Kinetic

correlates of methotrexate transport and therapeutic responsiveness in murine tumors. Cancer Res 1976; 36:1151-8.

7. Pratt WB, Ruddon RW, Ensminger WD, Maybaum J. Antimetabolites.. In: The anticancer drugs. Oxford University Press, 1994; 69-81

8. Kuriyama S, Matsumoto M, Mitoro A, et al. Electrochemotherapy for colorectal cancer with commonly used

chemotherapeutic agents in a mouse model. Digestive Diseases Sci 2000;

1568-77.

6

Addendum: At the beginning, we proposed the study topic as “The study of electrochemotherapy in squamous cell carcinoma and the related cell death mechanism” by using an animal model. However, the hairless mice with UV-induced squamous cell carcinoma established in the previous experiment (2000) began to die. Purchasing and proceeding new hairless mice and to induce squamous cell carcinoma on mouse skin need 5-6 months. We therefore decided to set up the in vitro model first as we shown here.

Cell lines

Voltage (V/cm)

IC₅₀

Bleomycin IC₅₀ Ratio

IC₅₀

Methotr exate IC₅₀ Ratio 90%

survival EP- EP+ EP-/EP+ EP- EP+ EP-/EP+

OECM-1 800 2.2x10^-4 7x10^-7 314.3 1x10^-7 5x10^-8 2

OC-2 1000 3.4x10^-4 4x10^-6 85 >1x10^-3 >1x10^-3 NA

SCC-15 550 2x10^-5 3.6x10^-7 55.6 1x10^-4 2.3x10^-5 4.4

Cal-27 1150 2.7x10^-4 2.8x10^-6 96.4 1.6 x10^-4 3.2 x10^-5 5

FaDu 1000 1.3x10^-4 1 x10-7 5012 4.5 x10^-6 3.6 x10^-6 1.3

BCC 900 1.4 x10^-4 2.5 x10-7 562 1 x10^-5 1 x10^-5 1

Colon 26 1024 1 x10^-4 1x10-6 100 6.3x10^-7 1x10^-7 6.3

B16f1 1000 1x10-4 2.3x10-7 434 >1x10^-3 >1x10^-3 NA

Table 1. OECM-1, OC-2, SCC-15, Cal-27: human oral cancer; FaDu: human hypopharyngeal carcinoma;

BCC: human basal cell carcinoma; Colon 26: murine colon cancer; B16F1: murine melanoma. NA:

non-applicable because the IC50 values were higher than the maximum concentration tested.

E le ctric field (V /cm )

0 5 0 0 1 0 0 0 1 5 0 0 2 0 0 0 2 5 0 0 3 0 0 0

Suvival %

0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0

F ig . 1 S u v iv a l o f B C C c e lls a fte r tre a tm e n t w ith e ig h t 9 9 u s p u lse s a t d iffe re n t fie ld s tre n g th s . E a c h p o in t re p re s e n ts th e m e a n o f th re e re p lic a te sa m p le s p e r e xp e rim e n t. B a rs re p re s e n t th e S E M .

7

Log MTX concentration (M)

-11 -10 -9 -8 -7 -6 -5 -4 -3 -2

Suvival %

0 10 20 30 40 50 60 70 80 90 100 110 120 130

EP- EP+

2C

Log Bleomycin concentration (M)

-11 -10 -9 -8 -7 -6 -5 -4 -3 -2

Suvival %

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140

EP- EP+

Figure 2. The cytotoxicity effects of methotrexate (MTX) or Bleomycin alone and combined with electric pulses. After electroporation, cells were cultured in medium containing folate (A). A 20-30% ehancement of cytotoxicity can be acheived when cultures cells in folate free medium (B). Effects of bleomycin on Colon 26 showing a larger decreases in IC50 when combined with electric pulses (C) regardless of the folate condition of the medium. EP:

electroporation.

Log MTX concentration (M)

-11 -10 -9 -8 -7 -6 -5 -4 -3 -2

Suvival %

0 10 20 30 40 50 60 70 80 90 100 110 120 130

EP- EP+

3A

Log MTX concentration (M)

-11 -10 -9 -8 -7 -6 -5 -4 -3 -2

Suvival %

0 10 20 30 40 50 60 70 80 90 100 110 120 130

EP- EP+

3B

Log MTX concentration (M)

-11 -10 -9 -8 -7 -6 -5 -4 -3 -2

Suvival%

0 10 20 30 40 50 60 70 80 90 100 110

120 EP-

EP+

Figure 3. A 10-30% enhancement of cytotoxicity can be found in all tested cell lines at low concentrations of methotrxate (10-7 to 10-10 M) when combined with electroporation. Representative plots of FaDu cells (A), and B16F1 (B) are shown here.