Bladder cancer is one of the most common malignant tumors which are threatening human's life and health seriously (Oosterlinck and Decaestecker, 2012). Surgery assisted by chemotherapy has become the main therapeutic schemes for bladder cancer. Reasonable chemotherapy could use the anti-cancer drugs cytotoxicity to the maximum, but bladder cancer was palindromic in the biological characteristics, and its relapse rate has been high. One of the reasons was the generation of anti-cancer drug resistance. Therefore, the resistance of anti-cancer drug has become a major obstacle in the chemotherapy of bladder cancer, and it must clarify the drug resistance mechanisms to eliminate the anti-cancer drug resistance of tumor cells. Like most malignant bladder cancer, it showed multidrug resistance (Multidrug Resistance, MDR) in anticancer drug resistance. Resistance mechanisms of tumor cells was quite complex, and summarized the followings: (1) drug transporting or uptaking barriers; (2) drug activation barriers; (3) target enzyme changes in the quantity and quality; (4) metabolic pathways increased; (5) decomposing enzyme increased; (6) repair mechanisms increased; (7) the cell membrane glycoprotein specific in excluding drugs increased; (8) DNA chains cross-linked reduced; (9) hormone receptor reduction or loss of function, and so on (Kunze et al., 2012).
The generation of multidrug resistance in tumor cells has become a major cause of failure in tumor chemotherapy, and also was the key problem which troubled in tumor therapy. More than 90% of tumor patients who died were affected by drug resistance in varying degrees according to the American Cancer Association. After the MDR discovery, many MDR mechanisms have been discovered successively. (Multidrug resistance gene 1, MDR-1) overexpression, topoisomerase decrease in the content or topoisomerase charges in character, the activity increase of glutathione-dependent detoxication enzyme system, the expression increase of the multidrug resistance-associated protein, and so on (Nordentoft et al., 2012).
Some report showed that some natural compounds could prevent the progression of cancer by modulating target molecular. Phenethyl isothiocyanate (PEITC) has been regarded as a tumor inhibitor and revealed the antitumor activity in many types of cancer, for example such as leukemia, prostate cancer, breast cancer, lung cancer, cervical cancer, colorectal cancer, etc. (Wu et al., 2010) Our study found that PEITC could overcome the ADM chemo-resistance in bladder cancer and we investigated the mechanism additionally.
MATERIALS AND METHODS
Cell Culture and Drug Dispensing
Human bladder carcinoma cell line T24 was obtained from the American Type Culture Collection. The cells are cultured in RPMI 1640 (Life Technologies) with 10% fetal bovine serum (FBS, Life Technologies) in incubator with 37°C, 5% CO2. Human ADM resistance bladder carcinoma cell line T24/ADM was established from our laboratory and cultured in RPMI 1640 (Life Technologies) with 10% fetal bovine serum (FBS, Life Technologies) and 0.01 mg/L ADM in incubator with 37°C, 5% CO2. PEITC (Sigma) were prepared in DMSO and diluted by RPMI 1640 without FBS, the final concentration of DMSO did not exceed five by ten thousand (v/v) which concentration would not induce cytotoxicity in cells (the assay and result not listed) and the 0µM PEITC treatment group of T24/ADM was control group. The cell proliferation inhibition ratio (%) = [1 − (OD value of treatment group/OD value of control group)] × 100.The linear range of maximum OD value of microplate reader that can be detected is 1.2 and minimus OD value is 0.1 though verification in our laboratory and the cell number optimization was performance to ensure result was accord with linear range.
PEITC and ADM Cytotoxicity Assay
Totally, 3 × 103 cells were seeded to the 96-well cell cultured plate and incubated overnight with 37°C, 5% CO2. Then different concentrations (0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100 µM) of PEITC were added to 96-well cell cultured plate and incubated for 24 hr. Cell Titer 96 Aqueous One Solution Reagent (Promega) was operated following to the manufacturer's instructions. Cell viability was determined by microplate reader (BioRad) at 490 nm.
A total of 3 × 103 T24 or T24/ADM cells were seeded to the 96-well cell cultured plate and incubated overnight with 37°C, 5% CO2. Then different concentrations (0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100 mg/L) of ADM were added to 96-well cell cultured plate and incubated for 24h. CellTiter 96 Aqueous One Solution Reagent (Promega) was operated following to the manufacturer's instructions. Cell viability was determined by microplate reader (BioRad) at 490 nm.
Drug Sensitivity Assay
Totally, 3×103 T24/ADM cells were seeded to the 96-well cell cultured plate and incubated overnight with 37°C, 5% CO2. Then different concentrations (0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100 mg/L) of ADM and 2 µM or 5µM PEITC were added to 96-well cell cultured plate and incubated for 24 hr. CellTiter 96 Aqueous One Solution Reagent (Promega) was operated following to the manufacturer's instructions. Cell viability was determined by microplate reader (BioRad) at 490 nm.
Cell Apoptosis Assay
A total of 3 × 105 cells were seeded to the 6-well cell cultured plate and incubated overnight with 37°C, 5% CO2. Then 0.05 mg/L ADM and 2 or 5 µM PEITC were added to 6-well cell cultured plate and incubated for 24 ht. Cells were harvested and washed twice with cold PBS, incubated with Annexin V-FITC/PI (BD) 30 min in dark. The apoptosis rate was analyzed by flow cytometry (BD) at 488 nm excitation and 620 nm emission.
Intracellular Rhodamine-123 Accumulation Assay
Totally, 3 × 105 cells were seeded to the 6-well cell cultured plate and incubated overnight with 37°C, 5% CO2. Then 2 or 5 µM PEITC were added to 6-well cell cultured plate and incubated for 24 hr. Cells were harvested and stained with 10 μM rhodamine-123 (Rh-123, Sigma) for 1 hr. The intracellular concentration of Rh-123 was determined by flow cytometry (BD) at 488 nm excitation and 525 nm emission.
Western Blotting Assay
A total of 3×105 cells were seeded to the 6-well cell cultured plate and incubated overnight with 37°C, 5% CO2. Then 2 or 5 µM PEITC were added to 6-well cell cultured plate and incubated for 24 hr. Cells were harvested and lysed with 1× modified RIPA buffer (50 mM Tris, 150 mM Nail, 1% Triton X-100, and 0.5% deoxycholate) containing 25 μg/mL leupeptin (Sigma), 10 μg/mL aprotinin (Sigma), 1 mM sodium orthovanadate, and 2 mM EDTA and extracted by centrifugation at 12,000g for 10 min in 4°C. The protein concentration of the samples was determined using a Bicinchoninic Acid Protein Assay Reagent kit (Qiagen), and the entire batches of cell lysates were analyzed using 12% SDS-PAGE. The samples were transferred to polyvinylidene difluoride membranes (PVDF, Millipore) and the membrane was blocked for 4 hr at room temperature with 5% nonfat milk, then incubated overnight at 4°C with primary antibody (Topo-II,1:500;MDR1,1:800; MRP1,1:800;bcl-2,1: 800; GST-π,1:500;NF-κB, 1:500;Survivin, 1:500;Twist, 1:500; PTEN, 1:500;p-Akt,1:800; p-JNK,1:800; Akt,1:800; JNK,1:800) (Santa Cruz). The membrane was washed with Tris-Buffered Saline (0.1%Tween 20) and incubated with peroxidase-labeled secondary antibodies at 1:2,000 diluted (Vector Laboratories) for 1 hr. Finally, membrane was exposed to the HRP substrate (Millipore) for 1 min and visualized. Total-Akt, total-JNK or β-actin (1:5,000) (Sigma) were used for loading control.
Real Time PCR Assay
A total of 3×105 cells were seeded to the 6-well cell cultured plate and incubated overnight with 37°C, 5% CO2. Then 2 or 5 µM PEITC were added to 6-well cell cultured plate and incubated for 24 hr. Cells were harvested and the total cellular RNA was isolated using Trizol reagent (Invitrogen Life Technologies, USA) based on the manufacturer's instructions. The cDNA pool for each cell line was synthesized using 1 μg of total RNA and SuperScript reverse transcriptase as described in the manufacturer's protocol (TaKaRa). The transcription level of target genes were detected by real-time PCR through icycler iQ detection system (Bio-Rad) and GAPDH was used for normalization. The PCR reaction was the condition: 2 min at 50°C, 5 min at 95°C, some cycles of 20 sec at 95°C, and 40 sec at 60°C.The details of target genes were following: Topo-II, Genbank accession Number: NM_001067, (foward) 5′-CTTGTACTGCAG ACCCACA-3′, (reverse) 5′-ATAATAGAAT CAAGGGAAT TCCCAA ACTCGA-3′; MDR1, Genbank accession Number: NM_000927, (foward) 5′-TGACATTTATTCAAAGTT AAAAGCA-3′, (reverse) 5′-TAGACACTTTATGCAAACA TTTCAA-3′; MRP1, Genbank accession Number: NM_ 004996, (foward) 5′-TCAGCCCTTCCTGACAAGCT-3′, (reverse) 5′-TCTCTGCTGCAGGAGGTCCG-3′; bcl-2, Genbank accession Number: NM_000633, (foward) 5′- GTCATGTGTGTGGAGAGCGT-3′, (reverse) 5′-GCCGTA CAGTTCCACAAAGG-3′; GST-π, Genbank accession Number: NM_145792, (foward) 5′-TGGGCATCTGAAGC CTTTTG-3′, (reverse) 5′-GATCTGGTCACCCACGATG AA-3′; NF-κB, Genbank accession Number: NM_003998, (foward) 5′- AAAGGTTAGGGTCAAGAT-3′, (reverse) 5′-TAGGAATTGCAGGTGTAT-3′; Survivin, Genbank accession Number: NM_001168, (foward) 5′-GCCCAGTGTTTC TTCTGCTT-3′, (reverse) 5′-CCGGACGAATGCTTTTTAT G-3′; Twist, Genbank accession Number: NM_000474, (foward) 5′-AGTCCGCAGTCTTACGAGGA-3′, (reverse) 5′-GCAGAGGTGTGAGGATGGT-3′; PTEN, Genbank accession Number: NM_000314, (forward) 5′-GGACGAA CTGGTGTAATGATATG-3′, (reverse) 5′-TCTACTGTTTTT GTGAAGTACAGC-3′; GAPDH, Genbank accession Number: NM_002046, (forward) 5′-ATGTTCGTCATGGGTGT GAACCA-3′, (reverse)5′-TGGCAGGTTTTTCTAGACGGC AG-3′.
Data were expressed as means ± standard deviation (SD.), statistical analysis was carried out using Student's t-test (two-tailed) by SPSS 17.0 and P < 0.05 were considered statistically significant. All experiments were duplicated three times.
PEITC Increased T24/ADM Cells Drug Sensitivity
The cytotoxicity assay showed that PEITC could inhibit to the tumor cells proliferation, but there was not significant cytotoxicity in 2 µM (cell proliferation inhibition ratio <5%) and there was low cytotoxicity in 5 µM (cell proliferation inhibition ratio < 15%) (Fig. 1A). The similar result was obtained though cell viability assay by Vi-CELL Cell Viability Analyzer (Beckman Coulter), the data was not displayed. Therefore, we selected the concentration of 2 µM and 5µM to explore the multidrug resistance reversal effect and mechanism of PEITC. The cytotoxicity assay also showed that there was an increased cell proliferation inhibition ratio of ADM in T24 parent cells with time dependence and T24/ADM cells and the IC50 of T24 parent cells is 0.053 ± 0.004 mg/L, the IC50 of T24/ADM cells is 1.214 ± 0.015 mg/L (Fig. 1B), and we selected the concentration of 0.05 mg/L of ADM to study in the research.
The drug sensitivity assay showed that there was an increased ADM sensitivity of T24/ADM after PEITC treatment. As shown in Fig. 2, we found that the IC50 of the PEITC treatment groups (0.667 ± 0.055 mg/L in 2 µM PEITC group and 0.358 ± 0.029 mg/L in 5 µM PEITC group) were lower than that of the control group. The resistance index (RI) of control group is 22.91, the 2 µM PEITC group is 12.58, and the 5 µM PEITC group is 6.75. The RI is calculated by IC50 of resistant cell/IC50 of sensitive cell.
PEITC Increased T24/ADM Cells Apoptosis Rate
Cell apoptosis assay showed that PEITC treatment resulted in an increase of apoptosis rate of T24/ADM cells. As shown in Fig. 3, we found that there was a higher apoptosis rate in the PEITC treatment groups than that of the control group. Took above results together, indicating that PEITC could reverse the ADM resistance of the T24/ADM cells.
PEITC Increased Intracellular Accumulation of Rhodamine-123 in T24/ADM Cells
Rhodamine-123 is a fluorescent compound which could reflect the intracellular drug accumulation potential. Our results showed that the cells which was treated by PEITC could contain more Rhodamine-123 fluorescent intensity, indicating there was a higher intracellular drug concentration in the PEITC treatment groups than that of the control group (Fig. 4).
PEITC Regulates the Expression of Multidrug Resistance Relative Gene
Multidrug resistance gene (MDR1), multidrug resistance-associated protein (MRP1), bcl-2, DNA topoisomerase II (Topo-II) and glutathione s transferase π (GST-π) are all involved in the tumor multidrug resistance. The western blotting assay showed that PEITC treatment could increase the expression of Topo-II, meanwhile, decrease the expression of MDR1, MRP1, bcl-2, and GST-π than that of the control group (Fig. 5A). At same time, real time PCR assay showed the same tendency on mRNA expression of above genes (Fig. 5B).
PEITC Regulates the Expression of Multidrug Resistance Relative Signal Pathway Molecules
NF-κB, Survivin, Twist, PTEN, p-Akt, and p-JNK are also all involved in the tumor multi-drug resistance. The western blotting assay showed that PEITC treatment could decrease the protein expression of NF-κB, Survivin, Twist and p-Akt, and increased the protein expression of PTEN and p-JNK in T24/ADM cells (Fig. 6A). At same time, real time PCR assay showed the same tendency on mRNA expression of NF-κB, Survivin, Twist, and PTEN (Fig. 6B).
We have known that MDR is the result of different factors which contribute in different mechanisms. High expression of P-glycoprotein (P-gp) corded with the multidrug resistance gene MDR1 was one of the MDR main reasons and the main target point in reversing MDR (Henríquez-Hernández et al., 2012). The study showed that when the tumor cells surface increased expression MDR1 significantly, the accumulation of drugs reduced in cells; when blocked the function of MDR1, the cell resistance would obviously reversed. Moreover, MDR1 could redistribute the intracellular drug, pump the drug out of the cell and reduce intracellular drug accumulation (Hoffmann et al., 2010; Rocco et al., 2012).
MRP was a member of the ABC transporter protein superfamily and that was different from MDR1 drug resistance spectrum (Mhawech et al., 2003), suggested that it might be related to glutathione transferase (Wang et al., 2011) which reduced intracellular drug concentration and made it be cell drug resistance by the transmembrane shift of glutathione (GSH). In addition, MRP can be used as a defense mechanism, the efflux of exogenous into the cells, and isolated drugs in cells to make the drug can not combine with the target, and thus lead to resistance indirectly (Roundhill and Burchill, 2012).
DNA topoisomerase II (Topo-II) was essential ribozyme for the eukaryotic cell survival, involved many aspects of the ribozyme metabolism, including DNA replication, mRNA processing, chromosome segregation, the gene recombinant transcription, DNA damage and repair, and so on (Kim et al., 2010). When used the chemotherapy drugs, chemotherapy drugs could catch aggregates, make the non-cocovalently bound to covalently bound. If Top-DNA aggregates reduced, it would lead to the emergence of drug resistance (Koren et al, 2003). Some reports considered that isothiocyanate could develop the cancer chemopreventive activity through inhibiting to phase I enzymes cytochrome P-450s, such as glutathione S transferases (GST) (Wu et al., 2009). According to the position in the cell they could be divided into five types α, μ, θ, π and membrane-bound microsome. The GST-π was the most closely related with malignancy, and it mainly distributed in the digestive tract, urinary system and the respiratory epithelium. It combinated GSH with a lot of electrophilic compound including the anticancer drugs by catalysis, and enhanced the water solubility, excreted more easily. It had the activity of peroxidase, and the reduction peroxides that anticancer drugs generated to innoxious substance, thus causing the cell chemotherapeutic drug resistance.
Our result showed that PEITC treatment could up-regulate intracellular drug accumulation of T24/ADM cells, which we considered it was one of the mechanism of PEITC reversed T24/ADM cells multidrug resistance. Multidrug resistance gene (MDR1), multidrug resistance-associated protein (MRP1), DNA topoisomerase II (Topo-II) and glutathione s transferase π (GST-π) were report to regulate intracellular drug accumulation, accordingly, we considered that PEITC could reverse T24/ADM cells multidrug resistance through increasing intracellular drug accumulation by regulating the expression of above genes.
Many anticancer drugs could eventually trigger the programmed cell death of tumor cells by interfering with the cell growth process of tumor, metabolism, proliferation, and would lead cells to apoptosis (Pinto-Leite et al., 2012). When the apoptosis mechanism and the anti-apoptotic mechanisms were imbalance, tumor cell apoptosis rate decreased obviously, and the tumor would be not sensitive to chemotherapy. In the regulatory genes of the found cell apoptosis, bcl-2 gene was one of the most important apoptosis inhibiting genes, and its increased expression could significantly inhibit tumor cell apoptosis and it could inhibit the apoptosis pathway of P53-dependent and P53-independent, thereby significantly prolonged the cell growth phase, and made the tumor be resistant. Clinical studies had confirmed: the chemotherapy response rates of bcl-2 protein-positive expression in bladder cancer patients group was significantly lower than the response rates of bcl-2 protein-negative patients group. After using antisense bcl-2 technology to reduce the bcl-2 protein expression in the cells of resistant strains, it was significantly more sensitivity to ADM. Survivin was widely expressed in the body embryos and malignant tumor tissues. And it was closely related to the malignant behaviors such as tumor tissue transfer, infiltration and chemical resistance and so on. Survivin inhibited the cells apoptosis by inhibiting the activity of caspase-3 and 7 zymogen. The study found that using survivin antisense oligonucleotide to inhibit the survivin expression of normal and tumor cells resulted that the polyploid production was increasing when the cell apoptosis was raising [Li et al., 2012; Weyhenmeyer et al., 2012; Ye et al., 2012; Zhang et al., 2012; Goldsmith et al., 2012). Our result showed that PEITC treatment could down-regulate bcl-2 expression and promote cell apoptosis, so we think that it is also the one of the mechanism of PEITC reversed T24/ADM cells multidrug resistance.
PI3K/AKT pathway played an important role in tumor multidrug resistance. PI3K/AKT pathway was an important cell transformation pathways and it involved in the formation of drug resistance. The studies showed that the expression of phosphorylated AKT had increased cancer cell resistance to chemotherapeutic drugs and inhibit the expression of phosphorylated AKT could reverse AKT-mediated resistance and that would be down-regulate the expression of MDR. PTEN is the first tumor-suppressor gene found so far and it was found that there was PTEN inactivation in the majority of human tumors. In recent years, it was reported that the anti-cancer drug cisplatin, doxorubicin, taxol and so on could induction of tumor cell apoptosis by enhancing PTEN inhibiting cancer activity which negatively regulates PI3K/AKT pathway. It also showed that the PTEN transfected tumor cells, and the excessive expressed PTEN cancer cells were more sensitive to the etoposide and adriamycin-induced deaths (Gallardo et al; 2012; Li et al., 2010; Mulholland et al.; 2012; Tzenaki et al.; 2012; Xu et al., 2012).
The growing number research not only had confirmed Twist was related to tumor metastasis, but also had provided a basis for Twist-mediated tumor resistance. It was discovered through the study of a variety of human cancer cell lines that the cell lines of Twist high expression was stronger in resistance to paclitaxel and vincristine drug-resistant than the cell lines of Twist low expression and it suggested that Akt was a downstream target in Twist regulating functions (Al-Azayzih et al., 2012; Chen et al., 2012; Kang et al., 2012; McCubrey et al., 2012). A large number of reports confirmed that induced resistance mechanisms of tumor cells could be related with MARK related signaling pathways which regulates various cellular activities, including cell survival/apoptosis and chemoresistance such as vincristine resistance in gastric cancer cells, gifitinib resistance in breast cancer, imatinib resistance in acute lymphoblastic leukemia cells, and so on, meanwhile, some reports showed that activating MAPK signal pathway is an effective way to reverse drug resistance (Derin et al., 2008; Guo et al., 2008; Suzuki et al., 2010). In this report, we found that phosphorylation level of JNK was enhance by PITC suggesting MAPK pathway is a potential target of PITC to reverse drug resistance in T24/ADM cells.
In this study, we know that PEITC treatment could decrease the protein expression of NF-κB, Survivin, Twist, and p-Akt, and increased the protein expression of PTEN and p-JNK in T24/ADM cells, therefore we considered that PEITC reveal the multidrug resistance reversal potential by regulating all the activities of Akt and JNK and transcription factors expression such as NF-κB, Survivin, Twist, and PTEN.
In conclusion, our study showed that PEITC could devote an important effect in ADM resistance reversal of bladder cancer T24/ADM cells and it may be as a potential treatment strategy in bladder cancer, especially for those with drug resistance bladder cancer patient.