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Synergistic effect of phototherapy and chemotherapy on bladder cancer cells
Journal of Photochemistry & Photobiology, B: Biology 193 (2019) 148–154
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Journal of Photochemistry & Photobiology, B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Synergistic effect of phototherapy and chemotherapy on bladder cancer cells a
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a
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Mehdi Shakibaie , Maryam Vaezjalali , Hashem Rafii-Tabar , Pezhman Sasanpour a b
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Department of Medical Physics and Biomedical Engineering, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran Department of Microbiology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
ARTICLE INFO
ABSTRACT
Keywords: Metabolic activity Blue light Apoptosis Drug resistance Phototherapy
Drug resistance as an important barrier to cancer treatment, has a close relation with alteration of cancer metabolism. Therefore, in this study the synergistic effect of phototherapy and chemotherapy were investigated on the bladder cancer cells viability. The cytotoxicity effect of blue light irradiation was measured by the MTT assay. Glucose consumption, lactate and ammonium formation were analyzed in the blue LED-irradiated cancer cells culture. Also, the expression of some genes involved in apoptosis and epithelial–mesenchymal transition was assessed using real-time PCR in comparison with the control group. The analysis of the results indicated that blue light irradiation inhibited the cell viability in a dose-dependent manner. Blue light irradiation decreased the cell viability by 7% and 19% (p < .05) in 5637 cells at doses of 8.7 J/cm2 and 17.5 J/cm2 in comparison with the control group respectively. Glucose consumption, lactate and ammonium formation diminished in the blue LED-irradiated 5637 cells in both doses. The real time PCR results indicated that the expression of Bax increased in blue light-irradiated cells. In addition, the cell cycle analysis showed that blue light irradiation arrested the bladder cancer in the G1 phase. Also, the effect of combination therapy on cancer cells was investigated in presence of blue light irradiation and cisplatin. The obtained results of the MTT assay indicated that blue light irradiation enhance the cytotoxicity effect of cisplatin on bladder cancer cells.
1. Introduction Bladder cancer is one of the widespread problems in men while it is the fourth most common cancer for men worldwide [1]. There has been different types of developed therapy methods including surgery [2], chemotherapy [3], radiation therapy [4], and photodynamic therapy [5]. In addition to the traditional methods, there is a significant trend in novel techniques. Several processes are involved in cancer progression such as metastasis and drug resistance. Since the epithelial–mesenchymal transition (EMT) has a key role in drug resistance and cancer invasion, targeting the EMT markers is a promising approach for overcoming these problems [6]. High-level expression of the E-cadherin and low-level expression of the fibronectin, vimentin, and N-cadherin suppress the EMT process. Moreover, high level of the fibronectin expression stimulates drug resistance in cancer cells through signaling pathway of the integrin β subunit interaction [7]. Expression of the Bax as a pro-apoptotic member of the Bcl-2 family is down-regulated in bladder cancer cells [8]. Consequently, the drugresistance occurs in this cancer followed by an increase in expression of the anti-apoptotic genes [9–11]. Another mechanism that cancer cells recruit to survive from cytotoxic effect of chemotropic agents is ⁎
metabolic reprogramming participated in proliferation, metastasis, invasive and drug resistance of cancer cells [12–14]. Variation of concentration of metabolites and enzymes involved in glycolysis pathway was observed in tumor cells during cancer progression [15,16]. Several investigations have focused on metabolic inhibition of cancer cells by designing new drugs that prevent the cells from energy acquisition [17]. Since the biological activation of irradiated cells undergoes changes in both in vitro and in vivo assessments, phototherapy has been known as an effective technique in treatment of the diseases. Irradiation triggers a series of cellular responses involved in proliferation and differentiation of human cells [18]. Transferring electromagnetic energy to the photoreceptor molecules such as cytochrome C and flavoproteins plays the main role in this process [19]. The biological effects of irradiation act in a dose-dependent manner and the cellular responses are depended on different cell types and imposed wavelengths [20]. Laser and light-emitting diodes are well-known as appropriate instruments in phototherapy. Since the effects of laser and light-emitting diodes irradiation on cellular responses are the same, implementing light-emitting diodes is expanding in phototherapy because of being cost effective and available [18,21]. Cytotoxic effect of blue light irradiation has been reported for colon cancer [22], human fibrosarcoma [23] and melanoma cells [24].
Corresponding author. E-mail address: [email protected] (P. Sasanpour).
https://doi.org/10.1016/j.jphotobiol.2019.02.004 Received 20 November 2018; Received in revised form 21 January 2019; Accepted 15 February 2019 Available online 18 February 2019 1011-1344/ © 2019 Elsevier B.V. All rights reserved.
Journal of Photochemistry & Photobiology, B: Biology 193 (2019) 148–154
M. Shakibaie, et al.
Inhibition of cancer cells proliferation happened at high dose of energy such as 11.3 J/cm2 at wavelength of 450 nm (Fibrosarchoma) [23], and 54 J/cm2 at wavelength of 465 nm (Colon cancer) [22]. Also, the cytotoxicity effect at higher wavelengths has been reported for 18, 36 and 54 J/cm2 at 808 nm (Glioblastoma) [25]. Decrease in expression of collagen, integrin and actin was observed in human cells exposed to blue light [26]. The blue irradiation diminished the B16 melanoma cells growth and increased the percentage of the G0/G1 and G2/M phase of melanoma cancer cells [27]. Also, migration and invasion were inhibited in the blue LED-irradiated human fibrosarcoma cells [23]. In this study, the viability and metabolic activity of bladder cancer cells were investigated throughout the period of cultivation. Also, the expression of putative genes involved in epithelial-to-mesenchymal process, metabolic reprograming and apoptosis was analyzed in the blue LED-irradiated bladder cancer cells.
concentrations through the culture time. Cell proliferation was determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. At the end of cultivation (96 h), 20 μl of the MTT solution (5 mg/ml in the PBS) was added to each well and incubated in a dark space for 4 h at 37 °C in a humidified atmosphere (95%) of 5% CO2. After aspirating the MTT solution, 200 μl of the DMSO was added to the culture wells. Subsequently, the optical density of the cells was measured by a multi-well scanning spectrophotometer, at wavelengths of 570 and 620 nm. Furthermore, cytotoxicity was evaluated by analyzing lactate dehydrogenase (LDH) activity of cancer cell lines cultured in 4-well plates. The LDH release from the cells was measured by determining the LDH activity in supernatants. Then, the cells were washed two times with the PBS followed by adding 200 μl of triton X-100 0.5% (w/v) into each well and the intercellular LDH activity of the lysed cells was measured in all experiments. Then, the LDH release percentage was calculated by Eq. (1):
2. Experimental Section
%LDH release =
2.1. Materials
LDH activity in sup erna tan t LDH activity in sup erna tan t + LDH activity in lysed cells × 100
Dimethylsulfoxide (DMSO), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were purchase from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's Modified Eagle's medium (DMEM), Fetal bovine serum (FBS), streptomycin, penicillin, and trypsin were purchased from GIBCO (Invitrogen™, Grand Island, USA).
(1)
2.6. Cell Cycling Analysis The treatment and control group were harvested from 4-well plates and precipitated by centrifuge at 90 g for 5 min separately at 96 h culture time. Cell pellets were re-suspended with 500 μl PBS and vortexed gently. The cell fixation was carried out by adding cell suspension slowly to 70% ethanol. Ethanol-fixed cells were stained and analyzed by flow cytometry (BD FACSCalibur Flow Cytometer, BD Biosciences, USA).
2.2. Biochemical Analysis of Samples Glucose, lactate and ammonium concentrations were measured enzymatically by the glucose, lactate and ammonium assay kits (Greiner Diagnostic GmbH, Bahlingen, Germany) respectively. Also, cytotoxic effect of light-emitting diode irradiation on cancer cells was measured by the lactate dehydrogenase (LDH) activity assay kit (Greiner Diagnostic GmbH, Bahlingen, Germany).
2.7. Real-time Polymerase Chain Reaction
2.3. Cell Culture and Media
After aspirating the media from 4-well plates, the cells were washed with the PBS twice. The cells were removed from the plates by using trypsin solution (0.25% w/v) after cultivation time of 96 h. The suspended cells were precipitated by centrifuge at 90 g for 5 min. Cell pellets were re-suspended with 200 μl PBS and kept at −80 °C for the RNA extraction. Total RNA was extracted by high pure RNA isolation kit (Roche Applied Science, Germany) according to the manufacturer's instruction and the RNA quality and quantity were measured using a Nanodrop spectrophotometer (Thermo Fisher Scientific). The synthesis of the cDNA from an RNA template was performed using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher scientific) according to the manufacturer's instruction. Primer sequences applied in real-time PCR are listed in Table S1 (see Supporting information). The qRT-PCR analysis was accomplished using the Rotor Gene 3000 Real-Time PCR Machine and the SYBR® Green Real-Time PCR Master Mix (Applied Biosystems). Relative quantification was performed using the comparative Ct (2-∆∆Ct) method.
All procedures were performed in accordance with the Shahid Beheshti University of Medical Sciences ethical guidelines. Ethical approval (IR.SBMU.MSP.REC.1397.147) was obtained from the Research Ethics Committee of the School of Medicine, Shahid Beheshti University of Medical Sciences. The human bladder cancer cell line 5637 was obtained from Pasteur institute of Iran (Tehran, Iran). The cells were cultured in two T-25 culture flasks separately containing basal medium comprised of the DMEM supplemented with 10% (v/v) FBS, 100 μg/ml streptomycin, 100 IU/ml penicillin. The cell culture flasks were maintained in a humidified incubator at 5% CO2 and 37 °C. 2.4. LED Irradiation The bladder cancer cells were seeded in 4-well plates at the density of 1 × 104 cells/cm2. After 24 h, the media were replaced with PBS at the exposure time. The plates exposed to light-emitting diodes at different doses of energy (8.7 and 17.5 J/cm2). Irradiation was carried out every 24 h for 120 h culture time. Samples were then taken every 24 h throughout the period of cultivation. Cancer cell lines that were not irradiated with the LED were used as the control group and maintained at the same conditions. Spectral characteristics of the blue LED is shown in Fig. S1 (see supporting information).
2.8. Statistical Analysis All experiments were carried out in duplicate. The data were calculated as the mean of two experiments using the Microsoft Excel 2016 software. All statistical calculations were performed using the SPSS software (version 16). Independent-samples t-test was used for statistical analysis, and a P-value of P < .05 was considered significant. The significance level for expression of target genes was analyzed with the REST 2009 software (V2.0.13).
2.5. Evaluation of Cytotoxicity The cells suspended in the basal medium were added at the same seeding density (5000 cells/well) in 96-well tissue culture plates. Irradiation was carried out in accordance with section 2.4. Also, the cytotoxicity effect of cisplatin on 5637 cells was analyzed at different 149
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Fig. 1. Cytotoxicity effect of blue light irradiation throughout the period of cultivation. (A) at dose of 8.7 J/cm2 (B) at dose of 17.5 J/cm2.
3. Results
3.3. Cell Cycle Analysis
3.1. Blue Light Irradiation Cytotoxicity
The cell cycle analysis in Fig. 6 indicates that the accumulation cells in the G1 phase is higher in the blue LED-irradiated cells in comparison with the control group. The percentage of cells in the G1 phase was 53.02% and 45.78% for irradiated cells and the control group respectively. In contrast, the S phase of cell cycle decreased from 38.57% in the control group to 29.33% after blue light irradiation.
The results in Fig. 1 compared with the control group demonstrates that blue light irradiation reduced the cancer cell viability in a dosedependent manner based on the MTT assay. Compared with the control group, blue light decreased the cell viability on 5637 bladder cancer cells by 7% and 19% (p < .05) at doses of 8.7 J/cm2 and 17.5 J/cm2 at 96 h culture time respectively and these inhibitory effects maintained at 120 h culture time. While no significant (p < .05) effect was observed in the blue LED- irradiated cells in comparison with the control group at 48 h and 72 h culture time at dose of 8.7 J/cm2. The morphology of blue light-irradiated 5637 cells is shown in fig. S3 (See supporting information). Also, the cytotoxicity of blue light was evaluated by measuring the LDH activity. Fig. 2 indicates that the percentage of the LDH release was increased by 67% (p < .05) in blue light irradiated cells at the dose of 17.5 J/cm2 in comparison with the control group.
3.4. Apoptosis and Epithelial–mesenchymal Transition The expression of some genes involved in apoptosis and epithelial–mesenchymal transition was analyzed using real time PCR. As shown in Fig. 7, the expression of the Bax, as a pro-apoptotic member of the Bcl-2 family, increased in the blue-LED-irradiated cells while there was no significant effect on expression of the Bcl-xl, as an anti-apoptotic member of the Bcl-2 family. Also, the down-regulation of fibronectin expression was obtained in 5637 cells treated with the blue LED irradiation in comparison with the control group while it had no significant effect (p < .05) on expression of vimentin, N-cadherin and E-cadherin.
3.2. Metabolic Activity
3.5. Drug-resistance
Glucose consumption was measured at different dose of energy through the culture time. Compared with the control group in Fig. 3, the energy dose of 17.5 J/cm2 shows a decrease of 16% in glucose consumption while this alteration in glucose consumption was not significant at the dose of 8.7 J/cm2 at the end of culture time. Byproducts formation analysis, lactate from glycolysis and ammonium from glutaminolysis pathway, by assay kits are shown in Figs. 4 and 5. The results indicate that blue light irradiation decreased the concentration of lactate and ammonium by15% and 12% at energy dose of 17.5 J/cm2 compared with the control group at 96 h culture time respectively. Decrease in the by-products formation was not significant (p < .05) at the energy dose of 8.7 J/cm2 at the same time.
The cytotoxicity effect of cisplatin on 5637 bladder cancer cells was measured by the MTT assay with an increasing trend in concentration through the culture time. The obtained results, as shown in Fig. S2, indicate that cisplatin decreased the cell viability in a dose-and timedependent manner so that the half maximal inhibitory concentration (IC50) values calculated for cisplatin were about 8, 4 and 0.8 μg/ml at 48, 72 and 96 h culture time respectively (See supporting information). The synergistic effect of blue light irradiation/ 0.4 μg/ml of cisplatin on cancer cell viability is shown in Fig. 8. The cell viability decreased by 12% in the blue LED- irradiated 5637 cells at energy dose of 8.7 J/cm2 in presence of cisplatin and this decrease in cell viability is continued at dose of 17.5 J/cm2 and 0.4 μg/ml of cisplatin by 29% (p < .05) in
Fig. 2. Blue light irradiation increase percentage of LDH release in 5637 cell culture. (A) at dose of 8.7 J/cm2 (B) at dose of 17.5 J/cm2. 150
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Fig. 3. Glucose consumption at energy dose of 8.7 J/cm2 and 17.5 J/cm2 at 96 h culture time (A) 8.7 J/cm2 (B) 17.5 J/cm2.
Fig. 4. Concentration of produced lactate at different energy doses at 96 h culture time (A) 8.7 J/cm2 (B) 17.5 J/cm2.
Fig. 5. Ammonium concentration analyzed in 5637 cell culture at energy dose of 8.7 J/cm2 at 96 h (A), 17.5 J/cm2 at 96 h (B).
comparison with cisplatin-treated cells without irradiation at 96 h culture time.
key role in acquisition of drug resistance [31,32]. As shown in Figs. 3, 4, blue light irradiation diminished glucose consumption and lactate formation in 5637 bladder cancer cells. Cancer cells convert pyruvate to lactic acid by lactate dehydrogenase-A enzyme more than normal cells. Secretion of lactate within the tumor microenvironment not only decreases the pH but also influence the immune escape and metastasis. Describing the pH of tumor microenvironment originated from lactate secretion inhibiting the activity of the NK cells [33]. The obtained results indicated that lactate formation in the blue LED-irradiated cells culture was less than in the control group. Cancer cells recruit aerobic glycolysis to survive against many types of treatment such as chemotherapy and ionizing radiation [13,34]. Consequently, increasing the glucose consumption and lactate formation are inevitable in many cancer cells. Many cancer treatments induce metabolic reprogramming, which challenges the effectiveness of the therapeutic approaches such as chemotherapy [35] and radiotherapy [36]. The blue LED-irradiation not only inhibited cell proliferation but also decreased glucose consumption and lactate formation. Also, cell cycle analysis shown in Fig. 6, demonstrated that blue light irradiation had the positive effect on cell cycle arresting.
4. Discussion Inhibition of cancer cell proliferation is a strategy to suppress tumor progression. Moreover anti-cancer drugs are focused on inducing apoptosis and hindering cell proliferation. Elevated intracellular levels of reactive oxygen species (ROS) can induce apoptosis and arrest cell cycle in tumor cells. Various experimental studies have indicated the contribution of blue light irradiation in the induction of the ROS production where the elevated ROS levels induced apoptosis in treated cells [19,22,28]. As shown in Fig. 1, blue light irradiation diminished cell viability in a dose-dependent manner. Metabolic reprograming is depended on regulation of the expression of genes for enzymes involved in glycolysis and oxidative phosphorylation [29]. These alterations in energy demand management are associated with cancer progression, metastasis and drug resistance [30]. High glycolysis rate in drug-resistance cells demonstrated that glucose not only is a vital carbon source for cell proliferation, but also it plays a 151
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Fig. 6. Cell cycle analysis of 5637 cells at energy dose of 17.5 J/cm2 at 96 h culture time, (A) Untreated cells as control group, (B) Blue LED-irradiated cells.
Epithelial-mesenchymal transition (EMT) is another process that plays a key role in drug resistance. Up-regulation of mesenchymal genes such as vimentin, fibronectin and N-cadherin followed by down-regulation of epithelial genes such as E-cadherin, occlude and the ZO-1 enhance phenotypic changes and promote metastasis in cancer cells [40,41]. Loss of E-cadherin expression is a primary sign of the EMT. Ionizing radiation destroys cancer cells but this treatment can induce the EMT in this population [42]. In opposite to ionizing radiation that acts as a double-edged sword in cancer therapy, the expression of Ecadherin in the blue LED-irradiated cells had no significant changes (p < .05). Synergistic effect of blue light/cisplatin on cytotoxicity shown in Fig. 8 confirm that blue light irradiation attenuated drug resistance in 5637 cells. Since both cisplatin and blue light irradiation induce ROS generation in cancer cells [19,28,43,44], it seems that cancer cells cannot mitigate this level of the ROS production.
Fig. 7. Expression of some genes involved in the EMT and apoptotic processes of blue LED-irradiated bladder cancer cells dose of 5.5 J/cm2 at 96 h culture time. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
5. Conclusion
In addition, it is well known that expression of some genes such as the Bax, Bcl-xl and fibronectin is involved in drug resistance of cancer cells [10,37,38]. Down-regulation of the Bax and up-regulation of fibronectin enhance cancer drug resistance [7,9,39]. Obtained results from real time PCR indicated that blue light irradiation increased the expression of the Bax compared with control group.
There are many reports on the treatment of cancer cells by ionizing irradiation. It is revealed that this treatment acts as a double-edged sword. Ionizing irradiation stimulated metastasis, metabolic reprograming and cancer stem cells phenotype. Moreover, treatment of cancer cells with ultraviolet irradiation induced expression of Snail as a main transcription factor in the EMT process [45]. In addition, the most
Fig. 8. Synergistic effect of blue light irradiation/cisplatin at dose of 8.7 J/cm2 (A), 17.5 J/cm2 (B) 96 h culture time. Untreated blue LED-irradiated cancer cells with cisplatin were regarded as control group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 152
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probable side effects of blue light irradiation therapy such as expression of the EMT markers and cancer metabolism have been evaluated. The results of our study demonstrated that blue light irradiation inhibited cell growth without any alteration in the EMT genes expression and metabolism. Also, this treatment increased the sensitivity to cisplatin and decreased the glycolysis rate in bladder cancer cells. Synergistic effect of blue light irradiation and chemotherapy will reduce the side effects of cisplatin by decreasing the recommended dose of drug. Cancer treatment with more than one type of therapy is known as combination therapy. Combination therapies can be helpful to overcome cancer drug-resistance. Since approximately 75% of bladder cancer are nonmuscle-invasive [1] and > 90% of urinary tract cancers are transitional cell carcinoma [46], the papillary lesion appear on the surface of tissue [47] where it could be easily exposed to the blue light irradiation. Also, there are some researches focused on utilizing the application of optical fibers in photodynamic therapy and treatment of bladder cancer [48–50]. Consequently, the obtained results of the blue LED-irradiation on bladder cancer cells could be examined by in vivo studies.
[17]
[18]
[19] [20] [21] [22] [23]
Acknowledgment
[24]
This article has been extracted from the thesis written by Mr. Mehdi Shakibaie in School of Medicine Shahid Beheshti University of Medical Sciences. (Registration No: M 139). The authors would like to thank Dr. M. Shalala (Pasteur Institute of Iran) for her valuable technical support on this project.
[25] [26] [27]
Appendix A. Supplementary data
[28]
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jphotobiol.2019.02.004.
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Journal of Photochemistry & Photobiology, B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Synergistic effect of phototherapy and chemotherapy on bladder cancer cells a
b
a
a,⁎
Mehdi Shakibaie , Maryam Vaezjalali , Hashem Rafii-Tabar , Pezhman Sasanpour a b
T
Department of Medical Physics and Biomedical Engineering, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran Department of Microbiology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
ARTICLE INFO
ABSTRACT
Keywords: Metabolic activity Blue light Apoptosis Drug resistance Phototherapy
Drug resistance as an important barrier to cancer treatment, has a close relation with alteration of cancer metabolism. Therefore, in this study the synergistic effect of phototherapy and chemotherapy were investigated on the bladder cancer cells viability. The cytotoxicity effect of blue light irradiation was measured by the MTT assay. Glucose consumption, lactate and ammonium formation were analyzed in the blue LED-irradiated cancer cells culture. Also, the expression of some genes involved in apoptosis and epithelial–mesenchymal transition was assessed using real-time PCR in comparison with the control group. The analysis of the results indicated that blue light irradiation inhibited the cell viability in a dose-dependent manner. Blue light irradiation decreased the cell viability by 7% and 19% (p < .05) in 5637 cells at doses of 8.7 J/cm2 and 17.5 J/cm2 in comparison with the control group respectively. Glucose consumption, lactate and ammonium formation diminished in the blue LED-irradiated 5637 cells in both doses. The real time PCR results indicated that the expression of Bax increased in blue light-irradiated cells. In addition, the cell cycle analysis showed that blue light irradiation arrested the bladder cancer in the G1 phase. Also, the effect of combination therapy on cancer cells was investigated in presence of blue light irradiation and cisplatin. The obtained results of the MTT assay indicated that blue light irradiation enhance the cytotoxicity effect of cisplatin on bladder cancer cells.
1. Introduction Bladder cancer is one of the widespread problems in men while it is the fourth most common cancer for men worldwide [1]. There has been different types of developed therapy methods including surgery [2], chemotherapy [3], radiation therapy [4], and photodynamic therapy [5]. In addition to the traditional methods, there is a significant trend in novel techniques. Several processes are involved in cancer progression such as metastasis and drug resistance. Since the epithelial–mesenchymal transition (EMT) has a key role in drug resistance and cancer invasion, targeting the EMT markers is a promising approach for overcoming these problems [6]. High-level expression of the E-cadherin and low-level expression of the fibronectin, vimentin, and N-cadherin suppress the EMT process. Moreover, high level of the fibronectin expression stimulates drug resistance in cancer cells through signaling pathway of the integrin β subunit interaction [7]. Expression of the Bax as a pro-apoptotic member of the Bcl-2 family is down-regulated in bladder cancer cells [8]. Consequently, the drugresistance occurs in this cancer followed by an increase in expression of the anti-apoptotic genes [9–11]. Another mechanism that cancer cells recruit to survive from cytotoxic effect of chemotropic agents is ⁎
metabolic reprogramming participated in proliferation, metastasis, invasive and drug resistance of cancer cells [12–14]. Variation of concentration of metabolites and enzymes involved in glycolysis pathway was observed in tumor cells during cancer progression [15,16]. Several investigations have focused on metabolic inhibition of cancer cells by designing new drugs that prevent the cells from energy acquisition [17]. Since the biological activation of irradiated cells undergoes changes in both in vitro and in vivo assessments, phototherapy has been known as an effective technique in treatment of the diseases. Irradiation triggers a series of cellular responses involved in proliferation and differentiation of human cells [18]. Transferring electromagnetic energy to the photoreceptor molecules such as cytochrome C and flavoproteins plays the main role in this process [19]. The biological effects of irradiation act in a dose-dependent manner and the cellular responses are depended on different cell types and imposed wavelengths [20]. Laser and light-emitting diodes are well-known as appropriate instruments in phototherapy. Since the effects of laser and light-emitting diodes irradiation on cellular responses are the same, implementing light-emitting diodes is expanding in phototherapy because of being cost effective and available [18,21]. Cytotoxic effect of blue light irradiation has been reported for colon cancer [22], human fibrosarcoma [23] and melanoma cells [24].
Corresponding author. E-mail address: [email protected] (P. Sasanpour).
https://doi.org/10.1016/j.jphotobiol.2019.02.004 Received 20 November 2018; Received in revised form 21 January 2019; Accepted 15 February 2019 Available online 18 February 2019 1011-1344/ © 2019 Elsevier B.V. All rights reserved.
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Inhibition of cancer cells proliferation happened at high dose of energy such as 11.3 J/cm2 at wavelength of 450 nm (Fibrosarchoma) [23], and 54 J/cm2 at wavelength of 465 nm (Colon cancer) [22]. Also, the cytotoxicity effect at higher wavelengths has been reported for 18, 36 and 54 J/cm2 at 808 nm (Glioblastoma) [25]. Decrease in expression of collagen, integrin and actin was observed in human cells exposed to blue light [26]. The blue irradiation diminished the B16 melanoma cells growth and increased the percentage of the G0/G1 and G2/M phase of melanoma cancer cells [27]. Also, migration and invasion were inhibited in the blue LED-irradiated human fibrosarcoma cells [23]. In this study, the viability and metabolic activity of bladder cancer cells were investigated throughout the period of cultivation. Also, the expression of putative genes involved in epithelial-to-mesenchymal process, metabolic reprograming and apoptosis was analyzed in the blue LED-irradiated bladder cancer cells.
concentrations through the culture time. Cell proliferation was determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. At the end of cultivation (96 h), 20 μl of the MTT solution (5 mg/ml in the PBS) was added to each well and incubated in a dark space for 4 h at 37 °C in a humidified atmosphere (95%) of 5% CO2. After aspirating the MTT solution, 200 μl of the DMSO was added to the culture wells. Subsequently, the optical density of the cells was measured by a multi-well scanning spectrophotometer, at wavelengths of 570 and 620 nm. Furthermore, cytotoxicity was evaluated by analyzing lactate dehydrogenase (LDH) activity of cancer cell lines cultured in 4-well plates. The LDH release from the cells was measured by determining the LDH activity in supernatants. Then, the cells were washed two times with the PBS followed by adding 200 μl of triton X-100 0.5% (w/v) into each well and the intercellular LDH activity of the lysed cells was measured in all experiments. Then, the LDH release percentage was calculated by Eq. (1):
2. Experimental Section
%LDH release =
2.1. Materials
LDH activity in sup erna tan t LDH activity in sup erna tan t + LDH activity in lysed cells × 100
Dimethylsulfoxide (DMSO), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were purchase from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's Modified Eagle's medium (DMEM), Fetal bovine serum (FBS), streptomycin, penicillin, and trypsin were purchased from GIBCO (Invitrogen™, Grand Island, USA).
(1)
2.6. Cell Cycling Analysis The treatment and control group were harvested from 4-well plates and precipitated by centrifuge at 90 g for 5 min separately at 96 h culture time. Cell pellets were re-suspended with 500 μl PBS and vortexed gently. The cell fixation was carried out by adding cell suspension slowly to 70% ethanol. Ethanol-fixed cells were stained and analyzed by flow cytometry (BD FACSCalibur Flow Cytometer, BD Biosciences, USA).
2.2. Biochemical Analysis of Samples Glucose, lactate and ammonium concentrations were measured enzymatically by the glucose, lactate and ammonium assay kits (Greiner Diagnostic GmbH, Bahlingen, Germany) respectively. Also, cytotoxic effect of light-emitting diode irradiation on cancer cells was measured by the lactate dehydrogenase (LDH) activity assay kit (Greiner Diagnostic GmbH, Bahlingen, Germany).
2.7. Real-time Polymerase Chain Reaction
2.3. Cell Culture and Media
After aspirating the media from 4-well plates, the cells were washed with the PBS twice. The cells were removed from the plates by using trypsin solution (0.25% w/v) after cultivation time of 96 h. The suspended cells were precipitated by centrifuge at 90 g for 5 min. Cell pellets were re-suspended with 200 μl PBS and kept at −80 °C for the RNA extraction. Total RNA was extracted by high pure RNA isolation kit (Roche Applied Science, Germany) according to the manufacturer's instruction and the RNA quality and quantity were measured using a Nanodrop spectrophotometer (Thermo Fisher Scientific). The synthesis of the cDNA from an RNA template was performed using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher scientific) according to the manufacturer's instruction. Primer sequences applied in real-time PCR are listed in Table S1 (see Supporting information). The qRT-PCR analysis was accomplished using the Rotor Gene 3000 Real-Time PCR Machine and the SYBR® Green Real-Time PCR Master Mix (Applied Biosystems). Relative quantification was performed using the comparative Ct (2-∆∆Ct) method.
All procedures were performed in accordance with the Shahid Beheshti University of Medical Sciences ethical guidelines. Ethical approval (IR.SBMU.MSP.REC.1397.147) was obtained from the Research Ethics Committee of the School of Medicine, Shahid Beheshti University of Medical Sciences. The human bladder cancer cell line 5637 was obtained from Pasteur institute of Iran (Tehran, Iran). The cells were cultured in two T-25 culture flasks separately containing basal medium comprised of the DMEM supplemented with 10% (v/v) FBS, 100 μg/ml streptomycin, 100 IU/ml penicillin. The cell culture flasks were maintained in a humidified incubator at 5% CO2 and 37 °C. 2.4. LED Irradiation The bladder cancer cells were seeded in 4-well plates at the density of 1 × 104 cells/cm2. After 24 h, the media were replaced with PBS at the exposure time. The plates exposed to light-emitting diodes at different doses of energy (8.7 and 17.5 J/cm2). Irradiation was carried out every 24 h for 120 h culture time. Samples were then taken every 24 h throughout the period of cultivation. Cancer cell lines that were not irradiated with the LED were used as the control group and maintained at the same conditions. Spectral characteristics of the blue LED is shown in Fig. S1 (see supporting information).
2.8. Statistical Analysis All experiments were carried out in duplicate. The data were calculated as the mean of two experiments using the Microsoft Excel 2016 software. All statistical calculations were performed using the SPSS software (version 16). Independent-samples t-test was used for statistical analysis, and a P-value of P < .05 was considered significant. The significance level for expression of target genes was analyzed with the REST 2009 software (V2.0.13).
2.5. Evaluation of Cytotoxicity The cells suspended in the basal medium were added at the same seeding density (5000 cells/well) in 96-well tissue culture plates. Irradiation was carried out in accordance with section 2.4. Also, the cytotoxicity effect of cisplatin on 5637 cells was analyzed at different 149
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Fig. 1. Cytotoxicity effect of blue light irradiation throughout the period of cultivation. (A) at dose of 8.7 J/cm2 (B) at dose of 17.5 J/cm2.
3. Results
3.3. Cell Cycle Analysis
3.1. Blue Light Irradiation Cytotoxicity
The cell cycle analysis in Fig. 6 indicates that the accumulation cells in the G1 phase is higher in the blue LED-irradiated cells in comparison with the control group. The percentage of cells in the G1 phase was 53.02% and 45.78% for irradiated cells and the control group respectively. In contrast, the S phase of cell cycle decreased from 38.57% in the control group to 29.33% after blue light irradiation.
The results in Fig. 1 compared with the control group demonstrates that blue light irradiation reduced the cancer cell viability in a dosedependent manner based on the MTT assay. Compared with the control group, blue light decreased the cell viability on 5637 bladder cancer cells by 7% and 19% (p < .05) at doses of 8.7 J/cm2 and 17.5 J/cm2 at 96 h culture time respectively and these inhibitory effects maintained at 120 h culture time. While no significant (p < .05) effect was observed in the blue LED- irradiated cells in comparison with the control group at 48 h and 72 h culture time at dose of 8.7 J/cm2. The morphology of blue light-irradiated 5637 cells is shown in fig. S3 (See supporting information). Also, the cytotoxicity of blue light was evaluated by measuring the LDH activity. Fig. 2 indicates that the percentage of the LDH release was increased by 67% (p < .05) in blue light irradiated cells at the dose of 17.5 J/cm2 in comparison with the control group.
3.4. Apoptosis and Epithelial–mesenchymal Transition The expression of some genes involved in apoptosis and epithelial–mesenchymal transition was analyzed using real time PCR. As shown in Fig. 7, the expression of the Bax, as a pro-apoptotic member of the Bcl-2 family, increased in the blue-LED-irradiated cells while there was no significant effect on expression of the Bcl-xl, as an anti-apoptotic member of the Bcl-2 family. Also, the down-regulation of fibronectin expression was obtained in 5637 cells treated with the blue LED irradiation in comparison with the control group while it had no significant effect (p < .05) on expression of vimentin, N-cadherin and E-cadherin.
3.2. Metabolic Activity
3.5. Drug-resistance
Glucose consumption was measured at different dose of energy through the culture time. Compared with the control group in Fig. 3, the energy dose of 17.5 J/cm2 shows a decrease of 16% in glucose consumption while this alteration in glucose consumption was not significant at the dose of 8.7 J/cm2 at the end of culture time. Byproducts formation analysis, lactate from glycolysis and ammonium from glutaminolysis pathway, by assay kits are shown in Figs. 4 and 5. The results indicate that blue light irradiation decreased the concentration of lactate and ammonium by15% and 12% at energy dose of 17.5 J/cm2 compared with the control group at 96 h culture time respectively. Decrease in the by-products formation was not significant (p < .05) at the energy dose of 8.7 J/cm2 at the same time.
The cytotoxicity effect of cisplatin on 5637 bladder cancer cells was measured by the MTT assay with an increasing trend in concentration through the culture time. The obtained results, as shown in Fig. S2, indicate that cisplatin decreased the cell viability in a dose-and timedependent manner so that the half maximal inhibitory concentration (IC50) values calculated for cisplatin were about 8, 4 and 0.8 μg/ml at 48, 72 and 96 h culture time respectively (See supporting information). The synergistic effect of blue light irradiation/ 0.4 μg/ml of cisplatin on cancer cell viability is shown in Fig. 8. The cell viability decreased by 12% in the blue LED- irradiated 5637 cells at energy dose of 8.7 J/cm2 in presence of cisplatin and this decrease in cell viability is continued at dose of 17.5 J/cm2 and 0.4 μg/ml of cisplatin by 29% (p < .05) in
Fig. 2. Blue light irradiation increase percentage of LDH release in 5637 cell culture. (A) at dose of 8.7 J/cm2 (B) at dose of 17.5 J/cm2. 150
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Fig. 3. Glucose consumption at energy dose of 8.7 J/cm2 and 17.5 J/cm2 at 96 h culture time (A) 8.7 J/cm2 (B) 17.5 J/cm2.
Fig. 4. Concentration of produced lactate at different energy doses at 96 h culture time (A) 8.7 J/cm2 (B) 17.5 J/cm2.
Fig. 5. Ammonium concentration analyzed in 5637 cell culture at energy dose of 8.7 J/cm2 at 96 h (A), 17.5 J/cm2 at 96 h (B).
comparison with cisplatin-treated cells without irradiation at 96 h culture time.
key role in acquisition of drug resistance [31,32]. As shown in Figs. 3, 4, blue light irradiation diminished glucose consumption and lactate formation in 5637 bladder cancer cells. Cancer cells convert pyruvate to lactic acid by lactate dehydrogenase-A enzyme more than normal cells. Secretion of lactate within the tumor microenvironment not only decreases the pH but also influence the immune escape and metastasis. Describing the pH of tumor microenvironment originated from lactate secretion inhibiting the activity of the NK cells [33]. The obtained results indicated that lactate formation in the blue LED-irradiated cells culture was less than in the control group. Cancer cells recruit aerobic glycolysis to survive against many types of treatment such as chemotherapy and ionizing radiation [13,34]. Consequently, increasing the glucose consumption and lactate formation are inevitable in many cancer cells. Many cancer treatments induce metabolic reprogramming, which challenges the effectiveness of the therapeutic approaches such as chemotherapy [35] and radiotherapy [36]. The blue LED-irradiation not only inhibited cell proliferation but also decreased glucose consumption and lactate formation. Also, cell cycle analysis shown in Fig. 6, demonstrated that blue light irradiation had the positive effect on cell cycle arresting.
4. Discussion Inhibition of cancer cell proliferation is a strategy to suppress tumor progression. Moreover anti-cancer drugs are focused on inducing apoptosis and hindering cell proliferation. Elevated intracellular levels of reactive oxygen species (ROS) can induce apoptosis and arrest cell cycle in tumor cells. Various experimental studies have indicated the contribution of blue light irradiation in the induction of the ROS production where the elevated ROS levels induced apoptosis in treated cells [19,22,28]. As shown in Fig. 1, blue light irradiation diminished cell viability in a dose-dependent manner. Metabolic reprograming is depended on regulation of the expression of genes for enzymes involved in glycolysis and oxidative phosphorylation [29]. These alterations in energy demand management are associated with cancer progression, metastasis and drug resistance [30]. High glycolysis rate in drug-resistance cells demonstrated that glucose not only is a vital carbon source for cell proliferation, but also it plays a 151
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Fig. 6. Cell cycle analysis of 5637 cells at energy dose of 17.5 J/cm2 at 96 h culture time, (A) Untreated cells as control group, (B) Blue LED-irradiated cells.
Epithelial-mesenchymal transition (EMT) is another process that plays a key role in drug resistance. Up-regulation of mesenchymal genes such as vimentin, fibronectin and N-cadherin followed by down-regulation of epithelial genes such as E-cadherin, occlude and the ZO-1 enhance phenotypic changes and promote metastasis in cancer cells [40,41]. Loss of E-cadherin expression is a primary sign of the EMT. Ionizing radiation destroys cancer cells but this treatment can induce the EMT in this population [42]. In opposite to ionizing radiation that acts as a double-edged sword in cancer therapy, the expression of Ecadherin in the blue LED-irradiated cells had no significant changes (p < .05). Synergistic effect of blue light/cisplatin on cytotoxicity shown in Fig. 8 confirm that blue light irradiation attenuated drug resistance in 5637 cells. Since both cisplatin and blue light irradiation induce ROS generation in cancer cells [19,28,43,44], it seems that cancer cells cannot mitigate this level of the ROS production.
Fig. 7. Expression of some genes involved in the EMT and apoptotic processes of blue LED-irradiated bladder cancer cells dose of 5.5 J/cm2 at 96 h culture time. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
5. Conclusion
In addition, it is well known that expression of some genes such as the Bax, Bcl-xl and fibronectin is involved in drug resistance of cancer cells [10,37,38]. Down-regulation of the Bax and up-regulation of fibronectin enhance cancer drug resistance [7,9,39]. Obtained results from real time PCR indicated that blue light irradiation increased the expression of the Bax compared with control group.
There are many reports on the treatment of cancer cells by ionizing irradiation. It is revealed that this treatment acts as a double-edged sword. Ionizing irradiation stimulated metastasis, metabolic reprograming and cancer stem cells phenotype. Moreover, treatment of cancer cells with ultraviolet irradiation induced expression of Snail as a main transcription factor in the EMT process [45]. In addition, the most
Fig. 8. Synergistic effect of blue light irradiation/cisplatin at dose of 8.7 J/cm2 (A), 17.5 J/cm2 (B) 96 h culture time. Untreated blue LED-irradiated cancer cells with cisplatin were regarded as control group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 152
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probable side effects of blue light irradiation therapy such as expression of the EMT markers and cancer metabolism have been evaluated. The results of our study demonstrated that blue light irradiation inhibited cell growth without any alteration in the EMT genes expression and metabolism. Also, this treatment increased the sensitivity to cisplatin and decreased the glycolysis rate in bladder cancer cells. Synergistic effect of blue light irradiation and chemotherapy will reduce the side effects of cisplatin by decreasing the recommended dose of drug. Cancer treatment with more than one type of therapy is known as combination therapy. Combination therapies can be helpful to overcome cancer drug-resistance. Since approximately 75% of bladder cancer are nonmuscle-invasive [1] and > 90% of urinary tract cancers are transitional cell carcinoma [46], the papillary lesion appear on the surface of tissue [47] where it could be easily exposed to the blue light irradiation. Also, there are some researches focused on utilizing the application of optical fibers in photodynamic therapy and treatment of bladder cancer [48–50]. Consequently, the obtained results of the blue LED-irradiation on bladder cancer cells could be examined by in vivo studies.
[17]
[18]
[19] [20] [21] [22] [23]
Acknowledgment
[24]
This article has been extracted from the thesis written by Mr. Mehdi Shakibaie in School of Medicine Shahid Beheshti University of Medical Sciences. (Registration No: M 139). The authors would like to thank Dr. M. Shalala (Pasteur Institute of Iran) for her valuable technical support on this project.
[25] [26] [27]
Appendix A. Supplementary data
[28]
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jphotobiol.2019.02.004.
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