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Functional Similarity of Anticancer Drugs by MTT Bioassay | OMICS International
ISSN: 1948-5956
Journal of Cancer Science & Therapy

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Functional Similarity of Anticancer Drugs by MTT Bioassay

Takaki Hiwasa1*,Takanobu Utsumi1, Mari Yasuraoka1, Nana Hanamura1, Hideaki Shimada2, Hiroshi Nakajima3, Motoo Kitagawa4, Yasuo Iwadate4,5, Ken-ichiro Goto6, Atsushi Takeda7, Kenzo Ohtsuka8, Hiroyoshi Ariga9 and Masaki Takiguchi1

1Department of Biochemistry, and Genetics, Chiba University, Graduate School of Medicine, Chuo-ku, Chiba 260-8670, Japan

2Department of Surgery, School of Medicine, Toho University, Ota-ku, Tokyo 143-8541, Japan

3Department of Molecular Genetics, Chiba University, Graduate School of Medicine, Chuo-ku, Chiba 260-8670, Japan

4Department of Molecular and Tumor Pathology, Chiba University, Graduate School of Medicine, Chuo-ku, Chiba 260-8670, Japan

5Department of Neurological Surgery, Chiba University, Graduate School of Medicine, Chuo-ku, Chiba 260-8670, Japan

6Department of Orthopaedic Surgery, National Hospital Organization, Shimoshizu Hospital, Yotsukaido, Chiba 284-0003, Japan

7Laboratory of Biochemistry, Graduate School of Nutritional Sciences, Sagami Women’s University, Sagamihara, Kanagawa 252-0383, Japan

8Laboratory of Cell & Stress Biology, Department of Environmental Biology, Chubu University, Matsumoto-cho, Kasugai, Aichi 487-8501, Japan

9Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060-0812, Japan

*Corresponding Author:
Dr. Takaki Hiwasa
Associate Professor, Department of Biochemistry and Genetics
Chiba University, Graduate School of Medicine
1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan
Tel: 81-43-226-2541
E-mail: hiwasa_ [email protected]

Received Date: July 11, 2011; Accepted Date: December 15, 2011; Published Date: December 17, 2011

Citation: Hiwasa T, Utsumi T, Yasuraoka M, Hanamura N, Shimada H, et al. (2011) Functional Similarity of Anticancer Drugs by MTT Bioassay. J Cancer Sci Ther 3: 250-255. doi: 10.4172/1948-5956.1000099

Copyright: © 2011 Hiwasa T, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

We prepared normal or Ha-ras-transformed NIH3T3 cells transfected stably or transiently with various tumorrelated genes. The chemosensitivity of the transfected clones to 16 anticancer drugs was compared to the parental control cells using the MTT assay. The chemosensitivity changes induced by transfected genes were calculated and expressed numerically as the Drug Chemosensitivity Index (DCI). High DCI values (indicating resistance) were frequently observed in cells expressing C/EBP?, C/EBP?, p53, p21, PTEN, dominant-negative MDM2, caspases, HSP90, COUP-TF1 and decorin. In contrast, transfectants expressing ras, src, erbB2 and calpastatin had low DCI values, indicating increased sensitivity. Thus, it may be possible to predict the sensitivity of cancer cells toward anticancer drugs based on the expression levels of these genes. We then performed a regression analysis of DCI values between anticancer drugs. The correlation coefficients (r) were relatively high between cisplatin, camptothecin, mitomycin C and etoposide, suggesting that the mechanisms of action of these drugs are similar. The r values of aclarubicin, vincristine, taxol and cytarabine were low, suggesting that each of these drugs has a different and unique effect. This analysis may provide a rationale for design of combination chemotherapy regimens.

Keywords

Anticancer drugs; Cancer chemotherapy; MTT assay

Abbreviations

ACR: Aclarubicin; AraC: Cytarabine; CDDP: Cisplatin; CPT: Camptothecin; DMEM: Dulbecco’s modified Eagle’s minimum essential medium; DMSO: dimethyl sulfoxide; 5-FU: 5-fluorouracil; HU: hydroxyurea; IFM: ifosfamide; MCNU: methyl 6-[3-(2-chloroethyl)-3-nitrosoureido]-6-deoxy-α-D-glucopyranoside; MIT: mitoxantrone; MMC: mitomycin C; MTT: 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide; MTX: methotrexate; PBS: phosphate-buffered saline; PEP: peplomycin; PMSF: phenylmethylsulfonyl fluoride; Taxol: paclitaxel; 6-TG: 6-thioguanine; VCR: vincristine; VP-16: etoposide

Introduction

Chemotherapy using anticancer drugs is a useful therapeutic method for cancer. Thus far, many effective anticancer drugs have been developed. However, due to the adverse effects of each drug, their application is limited. Thus, combination therapy using smaller amounts of multiple drugs has become more and more common.

The efficacy of anticancer drugs varies among patients. This may be explained by differences in gene expression. For example, overexpression of P-glycoprotein results in prominent resistance to many drugs such as vincristine, etoposide, and paclitaxel [1]. Comprehensive cDNA microarray analysis has been carried out for various cancers to examine alterations of gene expression [2-8].

In addition, a large diverse panel of cultured human tumor cell lines was tested for sensitivity to anticancer drugs [9]. Although this analysis was efficient to discriminate anticancer drug-responsive tumor cells, the genetic backgrounds of the cell lines were so variable that the precise action mechanism remained unclear. In the present study, we prepared NIH3T3 mouse fibroblasts transfected stably or transiently with varying tumor-related genes, and examined their chemosensitivity to anticancer drugs. Variations in action mechanisms may provide a rationale for combination chemotherapy.

Materials and Methods

Chemicals

Dulbecco’s modified Eagle’s minimum essential medium (DMEM), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 6-thioguanine (6-TG), cytarabine (AraC), hydroxyurea (HU) and were purchased from Sigma (St. Louis, MO). Mitomycin C (MMC), methotrexate (MTX), 5-fluorouracil (5-FU) and mitoxantrone (MIT) were purchased from Merck Biosciences (Darmstadt, Germany). Camptothecin (CPT) was purchased form Biomol Research Laboratories (Plymouth Meeting, PA). Paclitaxel (Taxol) was purchased from Alexis Corporation (Lausen, Switzerland). Dimethyl sulfoxide (DMSO), etoposide (VP-16), cisplatin (CDDP) and peplomycin (PEP) were ob- tained from Wako Pure Chemicals (Kyoto, Japan). Vincristine (VCR) and ifosfamide (IFM) were obtained from Shionogi Pharmaceutica Co. Ltd. (Osaka, Japan); methyl 6-[3-(2-chloroethyl)-3-nitrosoureido]-6- deoxy-α-D-glucopyranoside (MCNU) from Mitsubishi Welfarma Co. Ltd. (Osaka, Japan); and aclarubicin (ACR) from Astellas Pharma Inc. (Tokyo, Japan).

cDNA clones were purchased from Toyobo Biochemicals (Osaka, Japan), Guthrie Research Institute (Sayre, PA) or Open Biosystems (Huntsville, AL). Some clones were provided by isolators.

Cell lines and culture

NIH3T3 mouse fibroblasts and Ha-ras-transformed NIH3T3 cells (ras-NIH3T3) [10] were cultured in DMEM supplemented with 5% bovine serum and 100 μg/ml of kanamycin. Cells were transiently or stable transfected with cDNAs in eukaryotic expression vectors such as pcDNA3 and pME18S-FL3. To isolate stable transfectants, cells were transfected with the expression plasmids together with the neo gene using LipofectAMINE reagent (Life Technologies, Carlsbad, CA), and then selected by culture in the presence of G418 (400 μg/ml) for two weeks as described [11-13]. A total of 135 stable transfectants and 41 transiently transfected cells were examined for chemosensitivity toward 16 anticancer drugs.

Western-blotting and RT-PCR analyses

The protein expression levels of transfected genes were examined by Western blotting [12,13]. The cells were washed with phosphatebuffered saline (PBS) and lysed in 0.5% Nonidet P-40, 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 50 μM leupeptin, 50 μM antipain, 50 μM pepstatin A and 50 μM acetyl-Leucyl- Leucyl-norleucinal for 10 min at 4°C. The cell lysate was centrifuged at 13,000×g for 10 min and the supernatant was used as cytoplasmic cell extract. The pellet was used as the nuclear fraction. The samples were analyzed by Western blotting using antibodies purchased from Santa Cruz Biotechnology (Santa Cruz, CA), followed by detection using ImmunoStar Reagents (Wako Pure Chemicals).

When the commercial antibodies are not available, the mRNA expression levels were examined by RT-PCT (reverse transcriptionpolymerase chain reaction) as described previously [13,14]. Briefly, total cellular RNA was isolated from the tumor tissues using FastPure RNA Kit (Takara Biochemicals, Kyoto, Japan). Reverse transcription was carried out with oligo(dT)20 primer using the ThemoScript RT-PCR System (Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions. The mRNA expression levels were examined via quantitative realtime RT-PCR using the Universal Probe Library system (Roche, Basel, Switzerland).

Evaluation of cytotoxicity of anticancer drugs

Chemosensitivity of transfected cells was examined using the MTT assay according to the method of Mosmann [15] as previously described [16,17]. Cells at exponentially growing phase were used. Five thousand cells per well (100 μl) were plated in 96-well plates in the presence of various concentrations of anticancer drugs, and cultured for three days. The activity of mitochondrial succinic dehydrogenase was measured by incubation for 4 h in the presence of 0.5 mg/ml of MTT (3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide). Absorbance reflects the viable cell number and was measured at 570 nm with a reference wavelength of 650 nm using a microplate reader. Absorbance reflects the viable cell number and was expressed as a percentage of that of cells cultured in the absence of anticancer drugs.

For the combination treatment, ras-NIH3T3 were added with 5-FU (50 ng/ml), PEP (500 ng/ml) or MTX (50 ng/ml), and immediately after the addition, plated in the presence of varying concentrations of MIT, PEP or VP-16.

Data mining

The difference in the chemosensitivity between the parent and transfected cells was calculated and expressed numerically as the Drug Chemosensitivity Index (DCI) [17-19] as follows: DCI = log (IC40,transfectant/IC40,parent) (Figure 1).

cancer-science-therapy-MTT-assay

Figure 1: Representative results of the MTT assay. The relative cell viability was measured using the MTT assay. The concentration of drug is shown on the abcissa and the absorbance of the formazan, which represents the relative cell viability, is shown on the ordinate. Sensitivity curves of two transfectants, in addition to the parental ras-NIH3T3 cells, are shown. The positions of drug concentrations giving 40% inhibition in two transfectants (IC40,transfectant-1 and IC40,transfectant-2) and in the parental cells (IC40,parent) are shown. A dotted line indicates 60% (40% inhibition) of cell viability. Broken lines showed by arrows indicate the concentrations of IC40,transfectant-1, IC40,transfectant-2 and IC40,parent. DCI values were calculated and expressed numerically as follows: DCI = log (IC40,transfectant/IC40,parent)

Results and Discussion

Chemosensitivity to anticancer drugs of transfected cells

Mouse fibroblasts, NIH3T3 or ras-NIH3T3, were transfected with various cancer-related genes, and the chemosensitivity to 16 different anticancer drugs was examined by MTT assay. Table 1 summarizes the list of genes of which the transfection induced resistance or sensitization against each anticancer drugs. The DCI values reflect the extent how resistant of the transfected cells were converted by the gene transfection [17-19]. The full version of list of DCI values are shown in Supplementary Table S1. High DCI values (indicating drug resistance) were frequently observed in cells expressing C/EBPα, C/EBPβ, wildtype p53, p21, PTEN, mutated MDM2, caspases, HSP90, COUP-TFI and decorin. In contrast, transfectants expressing ras, src, erbB2, calpastatin, mutated p53 and wild-type MDM2 had low DCI values, indicating increased sensitivity. Thus, it may be possible to predict the sensitivity of cancer cells toward anticancer drugs based on the expression levels of these genes. It should be noted that oncogenes such as ras, src, erbB2 and MDM2 increased the chemosensitivity against some, if not all, anticancer drugs whereas tumor suppressor genes such as p53, p21 and PTEN reduced the sensitivity. This may justify the application of anticancer drugs for cancer therapy.

CDDP 5-FU MTX CPT
Transfected  genes Transfected  genes Transfected  genes Transfected  genes
Resistant Sensitive Resistant Sensitive Resistant Sensitive Resistant Sensitive
C/EBPa N-ras DAN Ha-ras C/EBPa v-Src DAN Ha-ras
C/EBPb Ha-ras C/EBPa TERT-WT DAN E2F HSP90 N-ras
C/EBPb PTEN-A3 Bax* v-Src Caspase-3 APP RhoA-DN Akt-DN
HSP90 HDAC1 Hsdj IkB-TDN PTEN-G129R Akt-DN C/EBPb ErbB2
Hsdj DJ1-WT Cystatin a Axin p53-WT Calpain 30K C/EBPb Calpastatin
v-Src PARK7 DnaJ Rac1 DnaJ cAMP-PK-CS Caspase-2 v-Src
COUP PTEN-G129R CyclinD1 PKCg-WT PKCa-KN ColXVIII MKRN1-mut Ki-ras
p21 Caspase-1 Caspase-3 Ki-ras Ras-N17 C/EBPa CBP TERT-WT
RhoA-DN APC N-ras STAT5A P/CAF WIG1 PDGF-RD HDAC1
FANCC CSK c-Myc R-Ras Decorin VDAC1 p16 FilaminA
               
VP-16 MIT MMC IFM
Transfected  genes Transfected  genes Transfected  genes Transfected  genes
Resistant Sensitive Resistant Sensitive Resistant Sensitive Resistant Sensitive
DAN DAP p53-WT MDM2-WT DAN N-ras C/EBPa Ras-N17
C/EBPb APC C/EBPb N-ras MKRN1-mut v-Src TERT-DN Akt-DN
PTEN-WT PARK7 CAPN10 TKT C/EBPa Ha-ras Hsp70 N-ras
PKCa-KN Ha-ras Bad PARK7 C/EBPb ErbB2 p53-mut Ki-ras
AISEC HIF1 PER3 HDAC1 C/EBPb Akt-DN HNF4 Ha-ras
IkB-TDN STAT4 c-Myc Ha-ras Caspase-2 p53 DnaJ Cystatin E
Bcl-2 STAT6 TERT-DN APC TERT-DN Ki-ras p16 Caspase-1
COUP OPRT Bcl-2 Cyclin D1 Caspase-3 TERT-WT C/EBPb v-Src
MDM2-mut HO COUP MSSP HSP90 Calpastatin MKRN1-mut WIG1
c-Myc DJ1-K130R KRas2-DN APC P/CAF Hsp70 C/EBPb p53
               
PEP ACR VCR Taxol
Transfected  genes Transfected  genes Transfected  genes Transfected  genes
Resistant Sensitive Resistant Sensitive Resistant Sensitive Resistant Sensitive
MKRN1-mut Ki-ras DAN WIG1 DAN OPRT APC Calpain 30K
p53-WT v-Src Caspase-2 PER2 p53-WT APC C/EBPb Axin
ARF1 Cystatin a p16 ColXVIII Decorin Rap1A Caspase-2 TSC1
HSP90 ErbB2 Bax Caspase-1 DLG FGFR-KR Decorin STAT2
Akt-DN N-ras Caspase-3 Ha-ras p21 Rac1 MDM2-WT p53-mut
PER3 PARK7 TERT-DN TK-1 C/EBPb APC DLG FilaminA
DAN FGFR-WT Bax RCC1 p16 b-Catenin Ki-ras Akt-DN
BH HDAC1 MM1 Ki-ras PER3 CRI1 Max STAT3
MDM2-WT Calpastatin TS Cystatin a PER-1 RAN PTEN-WT HNF4
Caspase-2 PIGPC1 Max ErbB2 Regucalcin c-Myc RhoA-DN Enigma
               
MCNU 6-TG AraC HU
Transfected  genes Transfected  genes Transfected  genes Transfected  genes
Resistant Sensitive Resistant Sensitive Resistant Sensitive Resistant Sensitive
C/EBPb Regucalcin Caspase-3 APP C/EBPb AMY1 DAN N-ras
Rab1A RAN C/EBPa PER2 MKRN1-mut Gluco-R Bcl-2 p21
RhoGDIα Akt-DN DAN ColXVIII OPRT Ki-ras m-Calpain ErbB2
ARF1 WIG1 CaMKIIa-CA CathL-mut Bcl-2 Ha-ras Caspase-3 HNF4
AISEC STMN Cystatina GUK1 AISEC Caspase-3 TERT-WT v-Src
MDM2-mut STAT6 HSP90 Calpastatin MDM2-WT FGFR-KR TSC1 Ki-ras
14-3-3z E2F C/EBPb RPA2 Decorin p53-mut PTEN-WT MDM2-WT
IKK-DN Caspase-3 PDGF-RD N-ras Bcl-XL RAN Abl STAT4
PKCa-KN C/EBPa HSP40 Ha-ras PKCa-KN Bax HSP90 PER-1
cAMP-PK-CS HSP40 HNF1 SDC1 Enigma HSP90 R-Ras CaMKIIa

Table 1: List of anticancer drug-sensitivity-related genes.

Regression analysis of DCI values

Because chemosensitivity was similar but not identical among anticancer drugs, we then performed a regression analysis of the DCI values shown in Table 1. The correlation coefficients (r) are summarized in Table 2. The r values were relatively high (significantly correlated) among CDDP, CPT, MMC and VP-16, suggesting that the mechanisms of action of these drugs are similar (Table 2, Figure 2). The r value between MMC and CPT was the highest. The r values of ACR, VCR, taxol and AraC were relatively low (no correlation), suggesting that each of these drugs has a different and unique effect.

.
  CDDP 5-FU MTX CPT VP-16 MIT MMC IFM PEP ACR VCR Taxol MCNU 6-TG AraC HU
CDDP   0.277 0.280 0.569 0.498 0.485 0.496 0.412 0.357 0.237 0.225 0.242 0.311 0.402 0.320 0.202
5-FU 0.278   0.510 0.438 0.393 0.094 0.466 0.329 0.235 0.404 0.241 0.134 0.109 0.382 0.115 0.306
MTX 0.281 0.510   0.484 0.426 0.275 0.515 0.355 0.407 0.350 0.358 0.180 0.173 0.523 0.229 0.356
CPT 0.568 0.438 0.484   0.509 0.473 0.767 0.501 0.496 0.417 0.344 0.258 0.297 0.588 0.257 0.539
VP-16 0.498 0.393 0.426 0.509   0.499 0.547 0.292 0.354 0.331 0.571 0.281 0.463 0.330 0.454 0.402
MIT 0.484 0.094 0.275 0.473 0.499   0.468 0.236 0.405 0.096 0.360 0.168 0.232 0.300 0.313 0.352
MMC 0.496 0.466 0.515 0.767 0.547 0.468   0.477 0.488 0.442 0.336 0.167 0.419 0.550 0.340 0.556
IFM 0.411 0.329 0.355 0.501 0.292 0.236 0.477   0.317 0.190 0.056 0.072 0.458 0.360 0.234 0.330
PEP 0.356 0.235 0.407 0.496 0.354 0.405 0.488 0.317   0.346 0.208 0.152 0.280 0.342 0.368 0.332
ACR 0.232 0.404 0.350 0.417 0.331 0.096 0.442 0.190 0.346   0.200 0.050 0.061 0.286 0.035 0.277
VCR 0.225 0.241 0.358 0.344 0.571 0.360 0.336 0.056 0.208 0.200   0.220 0.120 0.213 0.375 0.185
Taxol 0.242 0.134 0.180 0.258 0.281 0.168 0.167 0.072 0.152 0.050 0.220   0.402 0.219 0.057 0.151
MCNU 0.312 0.109 0.173 0.297 0.463 0.232 0.419 0.458 0.280 0.061 0.120 0.402   0.135 0.425 0.234
6-TG 0.402 0.382 0.523 0.588 0.330 0.300 0.550 0.360 0.342 0.286 0.213 0.219 0.135   0.168 0.394
AraC 0.320 0.115 0.229 0.257 0.454 0.313 0.340 0.234 0.368 0.035 0.375 0.057 0.425 0.168   0.257
HU 0.202 0.306 0.356 0.539 0.402 0.352 0.556 0.330 0.332 0.277 0.185 0.151 0.234 0.394 0.257  
 

Table 2: Correlation of DCI values between anticancer drugs.

cancer-science-therapy-Functional-similarity

Figure 2: Functional similarity between anticancer drugs. Combinations of two drugs with high r values are shown schematically. High r values suggest similar action mechanisms between drugs. Results in red indicate a closer relationship than results in black.

Combination chemotherapy is growing more common in cancer chemotherapy. For example, the combination of cyclophosphamide (IFM analogue), VCR and doxorubicin (ACR analogue) is effective for small cell lung cancer [20] and non-Hodgkin lymphoma [21]. The low r value among these drugs means that they works independently. Thus, additive effects can be expected. This was exemplified by the result that the high-r-value combination of MIT and PEP caused less cytotoxic effects than the low r value combination of MIT and 5-FU or MTX (Figure 3A).

cancer-science-therapy-combination-treatment

Figure 3: Results of combination treatment. ras-NIH3T3 cells were added with 5-FU (50 ng/ml), PEP (500 ng/ml) or MTX (50 ng/ml). Immediately after the addition, cells were plated in the presence of varying concentrations of MIT (A), PEP (B) and VP-16 (C). After culture for three days, the viabilities were measured by the MTT assay. The abscissa and ordinate represent the concentration of the latter drugs and the relative viability versus that in the absence of the latter drugs, respectively.

On the other hand, colon cancer and ovarian clear cell adenocarcinoma are treated with a combination of CPT and MMC [22,23], which showed a high r value (Table 2). Both CPT and MMC are effective toward ras- and erbB2-transfected cells (Table 1). Colon cancers are frequently accompanied by mutations in the Ki-ras gene [24,25]. Overexpression of erbB2 in ovarian carcinoma [26,27] may account for the sensitivity against CPT and MMC. Thus, if the target molecules are restricted, synergistic effects focused on the target can be expected. Furthermore, such high-r-value combinations may reduce the side effects caused by a high-dose application of a single drug. Likewise, the high r value combinations of PEP and MTX or VP-16 and MTX showed more suppressive effects than the low r value combinations of PEP and 5-FU or VP-16 and PEP (Figure 3B and C).

Consequently, this approach using regression analysis of DCI values of anticancer drugs may provide a theoretical basis for design of combination chemotherapy regimens.

Acknowledgements

Grant support

This work was partly supported by a Grant for 21st Century COE (Center of Excellence) Program and a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

We are grateful to Drs. Bert Vogelstein (Howard Hughes Medical Institute), Ygal Haupt (Weizmann Institute of Science), Julian Downward (Imperial Cancer Research Fund), Albert S. Baldwin, Jr. (Lineberger Comprehensive Cancer Center), Lucille A. Lascuola (Dana-Farber Cancer Institute), Daniel A. Bochar (Wistar Institute), Tadashi Akiyama (Waseda University), Tetsu Akiyama (Tokyo University), Sumio Sugano (Tokyo University), Eisuke Nishida (Kyoto University), Shigeo Ohno (Yokohama City University), Toshiharu Suzuki (Hokkaido University), Yu Katayose (Tohoku University), Koichi Suzuki (Toray Industries), Shigeru Sakiyama (Chiba Red Cross Blood Center), Masatoshi Maki (Nagoya University) and Kazumi Ishidoh (Tokushima Bunri University) for providing plasmids. We also thank Ms. Akiko Suganami, Masae Suzuki and Akiko Kimura for their helpful technical assistance.

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