Hyperthermia improves doxorubicin-based chemotherapy by activating mitochondrial apoptosis in bladder cancer

An-Chen Chang; Po-Chun Chen; Hung-En Chen; Te-Fu Tsai; Kuang-Yu Chou; Chao-Yen Ho; Thomas I-Sheng Hwang

doi:10.4103/UROS.UROS_6_22

ORIGINAL ARTICLE

Year : 2023 | Volume : 34 | Issue : 2 | Page : 64-69

Hyperthermia improves doxorubicin-based chemotherapy by activating mitochondrial apoptosis in bladder cancer

An-Chen Chang¹, Po-Chun Chen², Hung-En Chen³, Te-Fu Tsai⁴, Kuang-Yu Chou⁴, Chao-Yen Ho⁵, Thomas I-Sheng Hwang⁶
¹ Translational Medicine Center, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan
² Translational Medicine Center, Shin Kong Wu Ho-Su Memorial Hospital; Department of Life Science, National Taiwan Normal University, Taipei; Department of Medical Research, China Medical University Hospital, China Medical University, Taichung, Taiwan
³ Division of Urology, Department of Surgery, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan
⁴ Division of Urology, Department of Surgery, Shin Kong Wu Ho-Su Memorial Hospital, Taipei; Division of Urology, School of Medicine, Fu-Jen Catholic University, New Taipei City, Taiwan
⁵ Division of Urology, Department of Surgery, Shin Kong Wu Ho-Su Memorial Hospital; Institute of Traditional Medicine, School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
⁶ Division of Urology, Department of Surgery, Shin Kong Wu Ho-Su Memorial Hospital, Taipei; Division of Urology, School of Medicine, Fu-Jen Catholic University, New Taipei City; Department of Urology, Taipei Medical University, Taipei, Taiwan

Date of Submission	04-Jan-2022
Date of Decision	22-Mar-2022
Date of Acceptance	18-Apr-2022
Date of Web Publication	17-Jun-2023

Correspondence Address:
Thomas I-Sheng Hwang
Department of Surgery, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, 11102
Taiwan

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/UROS.UROS_6_22

Abstract

Purpose: Although intravesical chemotherapy has several antitumoral benefits, it can also have severe side effects. The development of novel therapeutic approaches for bladder cancer (BC) is thus warranted. Hyperthermia (HT) is a widely applicable adjuvant therapy in various cancers. Therefore, this study investigated the effect of HT on improving the chemosensitivity of BC. Materials and Methods: The BC cell lines 5637 and T24 were cultured and treated with HT (43°C) for 24 h. Then, cell viability and survival were assessed using resazurin reagent and colony formation assay, respectively. Western blot assay was used to analyze the levels of Bax, Bcl-2, cleaved caspase-3, and cleaved poly (ADP-ribose) polymerase (PARP) protein expression. Mitochondria degradation was observed by MitoTracker Green staining. Results: In BC cells, HT co-administered with various concentrations of doxorubicin significantly inhibited cell viability and survival. Moreover, HT combined with doxorubicin promoted mitochondrial apoptosis, which caused Bax upregulation and Bcl-2 downregulation. Levels of cleaved caspase-3 and PARP protein expression were also elevated after co-treatment. Conclusion: Taken together, HT improved the chemosensitivity of BC cells to doxorubicin. HT combined with chemotherapy further activated mitochondrial apoptosis in BC cells. The findings suggested that HT may serve as a potential adjunctive treatment for BC that is ready to be applied clinically.

Keywords: Apoptosis, bladder cancer, doxorubicin, hyperthermia, mitochondria

How to cite this article:
Chang AC, Chen PC, Chen HE, Tsai TF, Chou KY, Ho CY, Hwang TI. Hyperthermia improves doxorubicin-based chemotherapy by activating mitochondrial apoptosis in bladder cancer. Urol Sci 2023;34:64-9

How to cite this URL:
Chang AC, Chen PC, Chen HE, Tsai TF, Chou KY, Ho CY, Hwang TI. Hyperthermia improves doxorubicin-based chemotherapy by activating mitochondrial apoptosis in bladder cancer. Urol Sci [serial online] 2023 [cited 2023 Oct 2];34:64-9. Available from: https://www.e-urol-sci.com/text.asp?2023/34/2/64/378895

Introduction

Globally, bladder cancer (BC) is the fourth most common internal malignancy in men.^[1] In Taiwan, according to the Ministry of Health and Welfare, BC is the 8^th and 13^th most common tumor in men and women, respectively. Epidemiological studies have identified several risk factors for BC, including smoking, low daily water consumption, and workplace exposure to carcinogens.^[2] Current guidelines for the treatment of BC recommend transurethral resection of bladder tumors with intravesical Bacillus Calmette–Guérin (BCG) vaccine and intravesical chemotherapy, such as mitomycin C, doxorubicin, or epirubicin.^[3] However, there is ongoing clinical concern regarding the side effect burden of current therapies. Therefore, there is a clinical need for the development of safer therapeutic strategies for treating BC.

The concept of using hyperthermia (HT) to treat cancer has a history of more than 5000 years.^[4] The first record of using heat therapy for masses growing on the breast was in Ancient Egypt.^[5] HT is posited to have utility in the treatment of a variety of cancers, including head-and-neck cancer,^[6] rectal cancer,^[7] breast cancer,^[8] and BC.^[9] HT has previously been used as an adjuvant therapy in combination with surgery, chemotherapy, radiotherapy, and immunotherapy.^[10],[11] HT increases the radiosensitivity and chemosensitivity of cancer cells, effectively reduces tumor size, and improves the survival rate of patients.

Conventionally, the tumor and surrounding tissues are heated to establish HT in the range of 39°C–45°C by using heating equipment operated at a frequency of 13.56 or 8 MHz.^[12] High temperatures directly damage cancer cells and lead to apoptosis while causing minimal injury to normal cells.^[13] Apoptosis, a form of cell-intrinsic suicide program, is active during embryonic development and plays a crucial role in the maintenance of tissue homeostasis.^[14] Growing evidence suggests that HT-induced apoptosis is predominantly mediated by the generation of reactive oxygen species, which are produced by dysfunctional mitochondria.^[15] Furthermore, HT reduces the expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL, while increasing the expression of the pro-apoptotic protein Bax in HeLa cells.^[16] These results have contributed to increased knowledge of the role of HT in promoting pro-apoptotic conditions. However, the mechanisms underlying the efficacy of HT in treating BC have yet to be fully elucidated. The present study evaluated the hypothesis that HT increases chemosensitivity in BC cells through activation of mitochondrial apoptosis.

Our results demonstrate co-administration of HT, and the chemotherapy drug, doxorubicin, significantly decreased cell viability and increased cell death through activation of the mitochondrial apoptotic pathway. Combination treatment was associated with decreased expression of anti-apoptotic proteins Bcl-2 and increased cleavage of caspase-3 and poly (ADP-ribose) polymerase (PARP) compared with chemotherapy alone.

Materials and Methods

Ethics approval and informed consent statements

Not applicable. This study did not involve human subjects.

Cell culture

Human BC cell lines (5637 and T24) and normal epithelial cell lines (SV-HUC-1) were obtained from the Bioresource Collection and Research Center (Hsinchu, Taiwan). First, 5637, T24, and SV-HUC-1 cells were cultured in RPMI-1640, McCoy's 5A, and F12K medium, respectively. Culture media were supplemented with 10% fetal bovine serum, 2 mM GlutaMAX-1, 100 U/mL penicillin, and 100 μg/mL streptomycin. All cells were incubated at 37°C under 5% CO₂.

Co-administration of hyperthermia with doxorubicin

Human BC cell lines (5637 and T24) and normal epithelial cell lines (SV-HUC-1) were incubated at 43°C for 1 h and then exposed to various concentrations of doxorubicin (0–6.125 μg/mL) at 37°C for 24 h. After treatment, cell viability, colony formation, and apoptosis-related protein expression levels were measured.

Cell viability

Cells were seeded in 96-well plates before undergoing the above treatments. Cell viability was assayed using resazurin reagent according to the manufacturer's protocol (Biotium, Fremont, CA, USA). Fluorescent signals were measured with a 550-nm excitation filter and a 600-nm emission filter by using a Varioskan LUX multimode microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

Western blotting

Protein samples were resolved on sodium dodecyl sulfate–polyacryla1

mide gel electrophoresis and transferred to Immobilon polyvinyldifluoride membranes. Membranes were then blocked with protein-free blocking buffer (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at room temperature, followed by incubation with primary antibodies against Bax, Bcl-2, cleaved caspase-3, cleaved PARP, and β-actin (1:3000; GeneTex, Irvine, CA, USA) overnight at 4°C. After three washes with PBST, membranes were subsequently incubated with peroxidase-conjugated secondary antibodies (1:3000; GeneTex, Irvine, CA, USA) for 1 h at room temperature. Protein bands were visualized with enhanced chemiluminescence using Kodak X-OMAT LS film (Eastman Kodak, Rochester, NY, USA).

Colony formation assay

BC cells were seeded on six-well plates. The following day, cells were subjected to the following treatments. Colonies were allowed to grow for 7 days before being fixed and stained in 45% methanol (v/v), 45% dH₂O (v/v), 10% acetic acid (v/v), and 0.25% crystal violet (w/v), respectively. All images of colony formation were captured using a Ti2 microscopy system (Nikon, Tokyo, Japan).

Immunofluorescence of MitoTracker staining

BC cells were seeded on chamber slides and cultured as described above. Cells were then stained with MitoTracker Green (Thermo Fisher Scientific, Waltham, MA, USA) and counterstained with 4′,6-diamidino-2-phenylindole according to the manufacturer's protocol. Immunofluorescence images were acquired using the Nikon Ti2 microscopy system (Nikon, Tokyo, Japan).

Caspase-3 activity

BC cells were cultured as described above. BC cells were directly lysed through the addition of caspase-3 assay buffer containing (Z-DEVD)₂-R110 substrates and incubated at 37°C for 1 h. The fluorescence intensity of R110 was then measured with an excitation of 485 nm and emission of 535 nm by using the Varioskan LUX multimode microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

Statistics

All experiments were performed at least three times and each time in triplicate. Data were analyzed to detect statistically significant differences using Student's t-test to compare the means of two experimental groups, whereas one-way ANOVA followed by Bonferroni's post hoc comparison tests was used to compare the means of more than two groups. Results are the mean ± standard deviation. P < 0.05 was considered statistically significant.

Results

Hyperthermia increases doxorubicin-based chemosensitivity in bladder cancer cells

To induce HT, cells were incubated at 43°C for 60 min with functional assays performed after 24 h.^[17] The effect of HT and doxorubicin on cell viability was first assessed using a resazurin agent. As depicted in [Figure 1]a, [Figure 1]b, [Figure 1]c, BC cells were treated with various concentrations of doxorubicin (0–6.125 mg/mL) for 24 h. HT significantly reduced the viability of 5637 (Grade II) and T24 (Grade III) BC cells. In contrast, incubation with HT had no effect on sensitivity to doxorubicin in normal bladder epithelial cells (SV-HUC1).

Figure 1: Cell viability responses of bladder cancer cell lines to doxorubicin and HT. (a-c) A normal bladder epithelial cell line (SV-HUC-1) and bladder cancer cell lines (5637 and T24) were treated with (red) or without HT (black) for 1 h and then exposed to various concentrations of doxorubicin (0–6.125 μg/mL) for 24 h. After the treatment, resazurin-based cell viability was measured. Experiments were performed three times, and each time in triplicate. All values are the mean ± standard deviation compared with the control group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. HT: Hyperthermia

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Next, we performed colony formation assays to validate the effect of HT combined with doxorubicin on cell survival. As presented in [Figure 2]a, [Figure 2]b, [Figure 2]c, colony formation in 5637 and T24 BC cells was diminished following combination treatment. However, no difference in cell survival was observed in normal bladder epithelial cells treated with or without HT. Taken together, these results demonstrate that HT enhances chemosensitivity in BC cells, while normal cells have greater heat tolerance.

Figure 2: Effect of HT in combination with doxorubicin on the cell survival of BC cells. (a-c) A normal bladder epithelial cell line (SV-HUC-1) and bladder cancer cell lines (5637 and T24) were treated with (red) or without HT (black) for 1 h and then subjected to low concentrations of doxorubicin (0–0.2 μg/mL) for 24 h. The cell survival ability was detected using colony formation. Experiments were performed three times, and each time in triplicate. All values are the mean ± standard deviation compared with the control group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. BC: Bladder cancer, HT: Hyperthermia

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Hyperthermia synergized with doxorubicin to enhance mitochondrial degradation

Mitochondrial homeostasis involves biogenesis and degradation and plays a critical role in regulating cell survival.^[18] Research has indicated that HT promotes mitochondrial dysfunction and triggers apoptotic signals in cancer cells.^[15] Next, we investigated the effect of HT combined with doxorubicin on mitochondrial degradation using MitoTracker Green staining. A decrease in MitoTracker Green intensity indicates mitochondrial degradation and has been widely used to evaluate mitophagy.^[19] We found that HT in combination with doxorubicin significantly reduced the MitoTracker Green intensity of 5637 and T24 BC cells [Figure 3]a and [Figure 3]b. Taken together, these data suggest that mitochondrial degradation is involved in HT-enhanced chemosensitivity.

Figure 3: Effects of the combination of HT and doxorubicin on mitochondrial degradation. (a and b) Cancer cells (5637 and T24) were treated with or without HT for 1 h, followed by doxorubicin incubation for 24 h. After the indicated treatment, cells were stained with MitoTracker (green) and counterstained with DAPI (blue). Fluorescence images were captured using a Nikon Ti2 microscopy system. HT: Hyperthermia, DAPI: 4',6-diamidino-2-phenylindole

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Co-administration of hyperthermia and doxorubicin induces apoptotic cell death

Substantial mitochondrial degradation may induce cellular dysfunction and apoptosis. We therefore analyzed the effect of chemo–HT combination therapy on apoptotic signals. Bcl-2 protein is known to impair cell death, whereas Bax functions as a pro-apoptotic protein.^[20] We found co-administration of HT and doxorubicin synergistically reduced Bcl-2 protein expression and increased Bax protein expression [Figure 4]a and [Figure 4]b. Caspase-3, an important apoptotic protease, was further cleaved [Figure 4]c and [Figure 4]d and activated after treatment with HT in combination with doxorubicin [Figure 4]e and [Figure 4]f.

Figure 4: Additive effect of HT combined with doxorubicin-based chemotherapy on cell apoptosis. 5637 and T24 cells were treated with or without HT for 1 h, followed by doxorubicin stimulation for 24 h. (a-d) Levels of Bax, Bcl-2, and cleaved caspase-3 protein expression were measured using a Western blot assay. (e and f) Caspase-3 activity was analyzed using a Z-DEVD-R110 assay kit. Experiments were performed three times, and each time in triplicate. All values are the mean ± standard deviation compared with the control group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. HT: Hyperthermia

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Chemo–hyperthermia combination therapy and poly (ADP-ribose) polymerase cleavage-mediated regulation of apoptotic cell death

During cell apoptosis, activated caspase-3 cleaves the 116 kDa form of PARP into fragments of 89 and 24 kDa leading to induction of cell death.^[21] Therefore, PARP cleavage has been considered a hallmark of apoptosis.^[22] As illustrated in [Figure 5]a and [Figure 5]b, chemo–HT combination therapy significantly increased cleaved PARP protein expression. These results demonstrate that chemo–HT combination treatment results in greater induction of apoptotic signals compared with doxorubicin or HT treatment alone.

Figure 5: Effects of the combination of HT and doxorubicin on PARP cleavage. (a and b) 5637 and T24 cells were treated with or without HT for 1 h and then incubated with doxorubicin for 24 h. The levels of cleaved PARP protein expression were measured using a Western blot assay. PARP: Poly (ADP-ribose) polymerase, HT: Hyperthermia

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Discussion

HT has attracted increasing attention worldwide as a safe and effective medical modality for cancer treatment in addition to surgery, radiotherapy, chemotherapy, and immunotherapy.^[10] Although HT has utility in the clinical treatment of cancer, the molecular mechanisms underlying the effect of HT on the tumor environment remain unclear. In the present study, we demonstrate that HT increases the efficacy of chemotherapy by enhancing mitochondrial apoptosis in BC [Figure 6].

Figure 6: Schematic of the regulation of mitochondrial apoptosis by chemo–HT combination therapy. HT: Hyperthermia

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Chemotherapy has been a component of standard of care for patients with cancer, including BC, for many years.^[23] Chemotherapeutic agents for BC can be administered through a vein (systemic therapy) or directly into the bladder (intravesical therapy).^[24] However, chemotherapy-related side effects and chemotherapeutic resistance are of clinical concern. To address these limitations, the chemosensitizing activity of HT in cancers has been evaluated. A previous in vitro investigation reported that HT potentiates cisplatin and doxorubicin-reduced DNA repair and leads to elevated double-strand DNA breakage in breast cancer cells.^[25] The results of our study indicate that HT increases doxorubicin-induced mitochondrial degradation and apoptotic cell death in BC. Previous studies have demonstrated that co-administration of HT with doxorubicin induces cell cycle arrest, increases oxidative stress, and promotes apoptotic cell death in melanoma.^[26] HT also increases drug release from lysolecithin-containing thermosensitive liposome encapsulating doxorubicin (LTSL-DOX) in vitro and HT plus LTSL-DOX (HT-LTSL-DOX) treatment increases median tumor growth time in vivo.^[27] Moreover, HT-LTSL-DOX treatment leads to tumor microvascular damage, which in turn reduces the blood flow in tumor.^[28] Notably, Stein et al. demonstrated that HT promotes the nuclear translocation of Y-box-binding protein 1, which leads to enhanced expression levels of multidrug resistance (MDR)-associated genes such as MDR1 and multidrug resistance protein 1. However, this increased gene expression did not cause drug resistance following treatment with HT and Adriamycin. In contrast, combined treatment with HT and chemotherapy led to a significant decrease in cell survival by 35% in colon carcinoma.^[29] Further studies using patient samples are required to assess the risk of HT and chemotherapy inducing drug resistance genes. These results indicate that HT may influence various cellular functions to increase sensitivity to chemotherapy and overcome chemotherapeutic resistance.

Clinically, three-quarters of patients with BC are initially diagnosed as having nonmuscle-invasive BC (NMIBC); however, a previous study reported that approximately 50% of patients experience recurrence and progression to muscle-invasive BC.^[30] Notably, a meta-analysis of clinical data has suggested that chemo–HT combination therapy resulted in a 59% relative reduction in NMIBC recurrence compared with mitomycin C treatment alone.^[31] In another randomized controlled trial, Arends et al. observed higher recurrence-free survival after chemo–HT combination therapy compared to BCG alone.^[32] Furthermore, chemo–HT combination therapy appears to improve disease-free survival and bladder preservation rates.^[33]

The results of the present study demonstrate that HT induces mitochondrial apoptosis and thus enhances the sensitivity of BC cells to doxorubicin. Chemo–HT combination therapy may reduce the toxic side effects of doxorubicin and preserve patient quality of life. In summary, previous clinical evidence and our results indicate that HT has utility as an adjuvant therapy in BC with promising antitumor effects.

Conclusion

Collectively, our results demonstrate that HT enhances the sensitivity of BC cells to doxorubicin, which may reduce required dosages of doxorubicin and thus reduce doxorubicin-associated complications in clinical treatment. HT may have utility as a potential adjunctive treatment for BC.

Data availability statement

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Financial support and sponsorship

Nil.

Conflicts of interest

Prof. Thomas I-Sheng Hwang, an editorial board member at Urological Science, had no role in the peer review process of or decision to publish this article. The other authors decalared no conflicts of interest in writing this paper.

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