Extracorporeal shock waves effectively suppressed the proliferation and growth of colorectal cancer

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Extracorporeal shock waves effectively suppressed the proliferation and growth of colorectal cancer

https://doi.org/10.21203/rs.3.rs-4956573/v1

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Shock waves are widely used to treat various diseases and are garnering further attention for medical applications. Growing evidence suggests that the application of extracorporeal shock waves (ESV) could substantially inhibit tumor growth. However, the therapeutic efficacy of ESV in colorectal cancer and the underlying mechanisms remain elusive. Using colorectal cancer cell lines HT29 and SW620, we generated xenograft mouse models, and examined the therapeutic effect of a stepwise increase in ESV energy on tumor growth. In vivo, the application of 60 mJ ESV significantly delayed xenograft growth compared with 120 and 240 mJ ESV, with no impact on body weight or hepatic and renal function. Transcriptome analysis revealed that 60 mJ ESV suppressed colorectal cancer cell proliferation and induced cell apoptosis and ferroptosis; these findings were further confirmed by immunohistochemical staining and western blotting. Mechanistically, ESV suppressed cell proliferation and induced cell apoptosis and ferroptosis by activating the p53 signaling pathway, as evidenced in vitro study. In conclusion, we revealed that 60 mJ ESV could substantially inhibit colorectal cancer growth by activating p53 pathway-related proliferation inhibition and cell death. These findings suggest that ESV therapy could be a promising therapeutic strategy for colorectal cancer.

Physical sciences/Physics/Biological physics

Biological sciences/Cancer/Cancer therapy

Biological sciences/Cell biology/Cell death/Apoptosis

Biological sciences/Cell biology/Cell signalling

Health sciences/Gastroenterology/Gastrointestinal diseases/Gastrointestinal cancer/Colorectal cancer

extracorporeal shock waves

colorectal cancer

p53 signaling pathway

xenograft mouse models

ferroptosis

Colorectal cancer (CRC) is a prevalent malignancy of the digestive tract and has the third morbidity and the second highest mortality rate globally¹. Surgery is the most effective treatment for CRC, especially during the early tumor stages². However, a substantial of patients are diagnosed at an advanced disease stage, frequently after metastasis to distant sites in the body or are otherwise unresectable³. Chemotherapy and ionizing radiation are occasionally used to treat high-risk CRC⁴. However, these therapeutic regimens tend to have limited efficacy owing to their severe side effects and toxicity⁵. Moreover, chemotherapeutic drugs tend to indiscriminately kill cancer and normal cells. Despite the considerable advances in diagnosis and treatment, the mortality rate among patients with CRC remains high and poses a major health burden worldwide⁶. To improve the survival rate and prognosis of patients with CRC, there is an urgent need to explore safe and relatively efficacious therapeutic options.

Shock waves are supersonic pressure waves generated by shock tubes or lasers. Shock waves can deliver a sequence of transient pressure disturbances characterized by high amplitude, fast pressure rise, short pulse duration, and rapid propagation⁷. The shock wave technique is a non-invasive, targeted, and extracorporeal therapeutic strategy with numerous medical applications⁸. Shock wave therapy has been established as a tolerable and effective method for the disintegration of urinary calculi and treatment of chronic tendinitis, bone and wound healing, and ischemic heart disease, eliciting promising results ^7,9,10. Furthermore, shock waves can increase cell membrane permeability and have been successfully applied in drug delivery and gene transfer¹¹. However, the application of shock-wave therapy in oncology remains poorly explored. In pre-clinical models, shock waves, especially in combination with several antineoplastic drugs, were found to exert substantial effects. Shock waves are found to enhance the antineoplastic effect of molecular iodine supplements in breast cancer¹², and effectively suppress the proliferation and growth of tongue squamous cell carcinoma combined with 5-fluorouracil (5-FU)¹³. Approximately 30 years ago, shock waves were reported to increase the chemocytotoxicity of 5-FU in CRC cells by inducing cavitation¹⁴. However, research on the effects of shock waves in CRC has been limited to in vitro cytotoxicity and lacks comprehensive investigations into the underlying mechanisms. Moreover, the therapeutic effects of shock waves alone on CRC remain unknown.

p53, also known as the guardian of the genome, is a classic tumor suppressor that limits cellular proliferation by inducing cell cycle arrest, apoptosis and ferroptosis^15–17. p53 is activated by various stress signals, including DNA damage or oncogene activation, and orchestrates a plethora of downstream responses, such as DNA repair, cell cycle arrest, senescence, metabolism, and cell death^17,18. Several chemotherapy regimens and radiotherapy rely on p53 activation and its downstream responses to impede cancer cell growth. The activation of p53 for induction of p53-related outcomes, such as cell death in response to cancer therapy, has been explored to improve clinical outcomes^19,20. Therefore, identifying therapies capable of targeting p53 may be a promising direction for treating CRC. Accordingly, we speculate whether shock waves can activate p53-related cancer inhibition.

In the present study, we investigated the therapeutic effect of extracorporeal shock waves (ESV) alone in CRC using animal experiments. We further explored the potential molecular mechanisms of ESV-mediated inhibiting of CRC growth.

2.1 Impact of stepwise increased dosage of ESV energy on CRC xenograft tumor growth

To determine the therapeutic effect of ESV in vivo, we established a mouse model of subcutaneous tumors. Nude mice were subcutaneously injected with HT29 cells and observed daily for tumor growth (Fig. 1A). When the average tumor volume reached approximately 50 mm³, ESV therapy (60, 120, and 240mJ for 2000 shots) was administered every third day (Fig. 1B, Fig. S1). Body weight and tumor volume were measured at different time points (i.e., on days 0, 3, 6, and 9 after ESV therapy). There were no significant changes in body weight (Fig. 1C). In the control group, tumors grew by 262.8% on day 9. Administration of 60 mJ ESV significantly suppressed tumor growth, resulting in growth of only 141% (p<0.01). Administration of 120 mJ ESV generated a weak and non-significant inhibition, eliciting tumor growth of 197%, while 240 mJ ESV exerted no suppressive effect on tumor growth (249.3%, Fig. 1E). Upon examination of animals after the last treatment, animals in the 240 mJ ESV group exhibited bruises, suggesting significant vascular lesions (Fig. 1D). After screening for ESV energy in the CRC xenograft mouse, we selected 60 mJ for subsequent in vivo experiments.

2.2 Administration of 60 mJ ESV delayed the growth of CRC-derived xenografts

To further validate the therapeutic impact of 60 mJ ESV on CRC, we extended the experimental endpoint (i.e., by days 0, 3, 6, 9, 12, and 15 after ESV therapy), and examined the side effects using various methods. In the second set of experiments, the weight of mice was unaltered, consistent with the results of the previous experiment (Fig. 2A). Plasma alanine aminotransferase (ALT) and creatinine (Cr) levels are biomarkers of hepatic and renal function, respectively. Herein, there were no differences in serum ALT and Cr levels between the control and 60 mJ ESV groups, suggesting that 60 mJ ESV did not induce notable toxicity (Fig. 2B-C). Mice administered ESV had slower tumor growth than mice in the control group (Fig. 2D). At the experimental endpoint (day 15), mice in the 60 mJ ESV group had smaller tumors than mice in the control group (Fig. 2E). Pathological examination revealed the absence of notable abnormalities in the livers, indicating no liver metastasis (Fig. 2F). In addition, the administration of 60 mJ ESV suppressed the growth of SW620-derived xenografts, another colorectal cancer cell line (Fig. S2). Collectively, these results strongly suggested that 60 mJ ESV could effectively suppress CRC growth in vivo without inducing toxicity.

2.3 Administration of 60 mJ ESV blocks CRC cell proliferation

To elucidate the mechanisms through which shock waves inhibit CRC growth in transplant models, transcriptome analysis was performed using HT29-derived tumor tissues from the control (CON) and 60 mJ group. RNA-Seq captures the transcriptional information of cells in a fixed state and provides a snapshot of the DNA expression profile at a certain moment. RNA-Seq revealed that 60 mJ ESV reprogrammed the transcriptome, with 1748 upregulated genes and 2021 downregulated genes (Fig. 3A-B). Metascape was used to analyze differentially expressed genes between the two groups. As shown in Fig. 3C, downregulated differentially expressed genes were mainly enriched in “DNA metabolic process” and “mitotic cell cycle.” Mitosis is the basis for cell proliferation. Typically, tumor cells have abnormally active mitotic abilities and can proliferate rapidly. According to the RNA-seq data, administration of 60 mJ ESV could suppress the proliferation of CRC cells. Ki-67 staining was performed to evaluate cell proliferation using IHC staining. In the 60 mJ ESV group, the tumor tissues showed decreased Ki-67 staining (Fig. 3D, Fig. S3). These results indicated that ESV may exert a therapeutic effect by suppressing the proliferation of CRC cells.

2.4 ESV induces apoptosis and ferroptosis in CRC cells

Cell death and cell cycle arrest contribute to the inhibition of cell proliferation. To explore the mechanism through which ESV blocks CRC cell growth, we performed a Gene Set Enrichment Analysis (GSEA). Genes upregulated in the ESV group were enriched in pathways associated with apoptosis (Fig. 4A). Based on western blot analysis, the application of ESV elevated the expression of cleaved-PARP and cleaved-caspase3, both apoptosis biomarkers (Fig. 4B). IHC staining also verified the upregulated expression of cleaved-caspase3 in the ESV group (Fig. 4C, Fig. S3A). Moreover, the ferroptosis biomarkers, AKR1C1 and COX2, were increased in the ESV group than in the control group (Fig. 4D-E, Fig. S3B). These findings indicated that ESV could substantially induce apoptosis and ferroptosis in CRC tissues.

2.5 Effect of ESV on cell proliferation and death is related to the p53 pathway

To determine the potential pathway involved in ESV-mediated CRC inhibition, Metascape and GSEA analyses were applied to the RNA-seq data. The results revealed the enrichment of the p53 signaling pathway gene set in the ESV group (Fig. 5A-B). Given that the p53 signaling pathway is a classic tumor suppressor that plays an important role in tumor regulation, we speculated that ESV could inhibit CRC growth via this pathway. We detected the p53 mRNA and protein levels in HT29-derived tumors using real-time PCR and western blot, respectively. ESV-treated mice exhibited upregulated p53 expression when compared with control mice; this enhanced expression was confirmed by IHC staining for p53 (Fig. 5C-E, Fig. S3C). Taken together, these results strongly suggested that ESV could suppress cell proliferation and induce apoptosis and ferroptosis by upregulating p53 expression in vivo.

2.6 ESV inhibits proliferation and promotes death of CRC cells in vitro

In addition to the above in vivo animal experiments, we performed in vitro cell culture experiments. The CCK-8 assay demonstrated that 60 mJ ESV markedly suppressed the proliferation of HT29 cells in vitro (Fig. 6A). Flow cytometric analysis revealed that ESV-treated CRC cells had a greater proportion of cells in the G0/G1 phase (77.3%) than control cells (60.6%). ESV-treated cells were arrested at the G0/G1 phase of the cell cycle, and the percentage of cells in the S phase was reduced (Fig. 6B). Furthermore, flow cytometric analysis showed that treatment with ESV significantly increased the percentage of dead HT29 cells (PI⁺ cells, Fig. 6C). Treatment with ESV increased the levels of P53 protein in vitro, along with those of cleaved-PARP, cleaved-caspase3, AKR1C1 and COX2 (Fig. 6D). Collectively, these findings indicated that ESV could impede CRC growth via p53 pathway-mediated cell cycle arrest and cell death, both in vivo and in vitro.

Herein, we investigated the therapeutic effects of ESV monotherapy on blocking CRC growth both in vivo and in vitro, which elicited notable preclinical implications. First, animal experiments revealed that the administration of 60 mJ ESV decreased tumor growth without affecting body weight or liver and kidney functions. Second, in vivo and in vitro studies revealed that the administration of ESV not only suppressed CRC cell proliferation but also induced cell apoptosis and ferroptosis. Third, ESV therapy could exert tumor-suppressive effect by reactivating the p53 signaling pathway. These results strongly indicate that ESV therapy may be an effective therapeutic modality for suppressing CRC growth.

Currently, effective and safe methods to treat patients with advanced-stage or unresectable cancer are lacking²¹. ESV, which is non-invasive and targeted, could be valuable for tumor treatment. Therefore, we evaluated the therapeutic effects of ESV in CRC inhibition. The most important finding of this study was that administration of ESV alone could significantly reduce the tumor volume in nude mice with CRC transplanted subcutaneously. However, colorectal tumors are located in the abdominal cavity rather than on the body surface. Further studies exploring the effects of ESV therapy using spontaneous CRC models are needed, for example, the APC^min/+ mouse or azoxymethane/dextran sulfate sodium-induced mouse colitis-associated carcinogenesis model²². Moreover, the characteristics of the ESV can best be applied to relatively superficial tissues and organs. ESV was found to effectively suppress the proliferation and growth of tongue squamous cell carcinoma and breast cancer, which can be considered relatively superficial types of cancer in the human body^12,13. Importantly, specific instruments to administer shock-wave therapy in patients with breast cancer have been developed and are undergoing preliminary clinical trials.

As early as 30 years ago, scientists explored the potential of shock waves for tumor treatment. Shock waves can shatter tumor cells and injure the microvasculature, raising concerns regarding the risk of metastasis^23,24. Subsequent studies revealed that the effect of shock waves on tumors depended on the shockwave energy. Low-dose shock waves were shown to enhance free radical generation, ATP release, or membrane instability, which could induce apoptotic mechanisms, ultimately reducing tumor growth and diminishing the potential for tumor metastasis^12,25. Consistently, our study investigated three different shock wave energies, demonstrating that low-energy (60 mJ) shock waves could remarkably block CRC growth with no liver metastasis. In contrast, 240 mJ not only failed to inhibit tumor growth but also caused hematoma.

In addition to energy, other mechanical parameters of shock waves, including peak pressure, rise time, and shock wave impulse, can also impact therapeutic outcomes. Schmidt et al. conducted in vitro experiments to examine the effects of shock wave on U87 brain cancer cells. The authors found that when the incident peak pressure exceeded a lethal level, shock waves could induce substantial cell damage²⁶. Liao et al. found that shock waves with higher impulses led to decreased cell viability, whereas shock waves with similar peak pressures exerted distinct effects on cell viability²⁷. Herein, we specifically focused on the effects of shock waves of different energies on CRC growth. Additional studies are needed to investigate the effects of the other mechanical characteristics of shock waves on CRC cell viability.

In conclusion, we present new evidence demonstrating a novel method for treating CRC both in vivo and in vitro. The use of 60 mJ ESV could markedly suppress CRC growth by blocking the proliferation of cancer cells and inducing apoptosis and ferroptosis. Mechanistically, ESV may activate the p53 signaling pathway to inhibit CRC growth. Our study highlights that appropriate ESV energy may serve as an accessory option (i.e., “give it a try”) in those patients with terminal-stage CRC who are refractory to conventional therapy.

All experimental methods and protocols were performed in accordance with the relevant guidelines and regulations of the Beijing Neurosurgical Institute, and the protocols for animal procedures were approved by the ethics committee of Beijing Neurosurgical Institute Ethics Committee of Capital Medical University (BNI202304002).

4.1 Mouse Xenograft Studies

Six-to-eight-week-old male BALB/c nude mice were subcutaneously injected with 1 × 10⁶ HT29 in 100 µL. The mice were observed daily for tumor growth. When the average tumor volume reached approximately 50 mm³, the mice were divided randomly into four groups, including control (Ctrl), 60mJ, 120mJ, and 240mJ shock waves groups. The control group without any treatment, shock waves groups accepted ESV therapy with stepwise increased energy (60, 120, 240 mJ, 2000 shots, 12Hz) at days 0, 3, 6, 9 and 12 after therapy initiation. At the same time, the tumor volumes were measured once every 3 days to determine the effect of each therapeutic regimen. The lengths (L) and widths (W) of the tumors were measured, and the tumor volumes were calculated as (L × W²)/2.

4.2 ESV application and definition

The ESV machine, called lattice shock wave physiotherapy instrument (J Gendica, China), was utilized in the present study. In detail, the ESV energy could be adjusted prior to applying for the in vitro and in vivo studies. After a determination of machine parameters, the machine was switched on. The automatic shots to the specific tissues/cells were started on. We defined the ESV energy applied to animals as 60mJ, 120mJ, and 240mJ (2000 shots, 12Hz). Additionally, we defined the ESV energy applied to HT29 cell line as 60mJ, 2000 shots, 12Hz.

4.3 RNA Sequencing

RNA samples were prepared from three biological replicates of HT29-derived xenograft tissues using TRIzol reagent. RNA quality was quantified using RNA integrity number. Sequencing libraries were constructed using the NEB Next Ultra RNA Library Prep Kit for Illumina and quantified using the KAPA Library Quantification kit (KAPA Biosystems). The libraries were then sequenced on an Illumina HiSeq sequencer. 150bp paired-end reads were mapped to human hg19 genome. Cuffdiff was used to analyze differentially expressed genes between the samples.

4.4 Immunohistochemistry (IHC) analysis

Paraffin-embedded xenografts were cut into 4 mm sections. The sections were dewaxed and hydrated, and then blocked and incubated with primary antibodies against Ki67 (Cell Signaling Technology, 12202, 1:200), P53 (Proteintech, 60283-2-Ig, 1:1000), cleaved-caspase3 (Cell Signaling Technology, 9664T, 1:1000) at 4°C overnight. The signals were visualized using a two-step plus®Poly-HRP Anti-Mouse/Rabbit IgG Detection System (OriGene, PV9000). Counterstaining was performed using hematoxylin. The sections were digitally scanned under a microscope with an FL-20 cooled camera, and images were analyzed using the Mosaic™ V2.1 software.

4.5 Reverse Transcription-Quantitative Polymerase Chain Reaction

The total RNA content was isolated from cells or the tumor using TRIzol™ reagent (Invitrogen), treated with DNase I (Thermo Fisher Scientific, Cat# EN0521), and then reverse transcribed into cDNA using a Maximal First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Cat# K1622). Real-time polymerase chain reaction (PCR) was performed using an S1000 PCR instrument (Bio-Rad, Hercules, CA, USA) to quantify gene expression. The PCR data were normalized to glyceraldehyde-3-phosphate dehydrogenase expression at the mRNA level. The primers used are listed in Supplementary Table S1.

4.6 Western blotting

Proteins were extracted from tumor tissues or cells with RIPA buffer (0.01% EDTA, 0.1% TritonX-100 and 10% proteinase inhibitor cocktail). Protein concentrations were quantified with a protein assay kit (Bio-Rad). 100 µg lysate were separated on 10% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes. The membranes were probed overnight at 4°C with primary antibody against human P53 (Cell Signaling Technology, 48818S, 1:1000), β-Actin (Sigma, 1:10000), cleaved-caspase3 (Cell Signaling Technology, 9664T, 1:1000), cleaved-PARP (Cell Signaling Technology, 9541T, 1:1000), AKR1C1 (Abcam, ab192785, 1:1000), COX2 (Abcam, ab179800, 1:1000), followed by incubation with peroxidase-conjugated secondary antibody (Cell Signaling Technology, 1:10000) for 1.5 hours. The signal was visualized with ECL (Millipore).

4.7 Statistical analysis

All experiments were independently repeated at least three times unless stated otherwise. Statistical analyses were performed using the GraphPad Prism software (version 8.0). Data are presented as the mean ± standard deviation (SD). Two groups were compared using the Student’s t-test for unpaired data. Comparisons among more than two groups were performed using ANOVA and Tukey’s test. For all tests, P values ≤ .05, were considered statistically significant ( ^∗P < .05 ).

Funding

This work was supported by the National Nature Science Foundation of China (82203229); the Shenzhen Science and Technology Program (JCYJ20220531094005012), and the opening project of State Key Laboratory of Explosion Science and Technology (Beijing Institute of Technology, KFJJ22-16M).

Contributions

XLZ and QZS performed the experiments. XLZ and CR designed experimental protocols, interpreted data obtained from the experiments, and wrote the manuscript. GQL edited the manuscript.

Data availability

All raw data used to generate the results of this study are available from the corresponding author by request.

Competing interests statement

The authors declare no competing interests.

Ethic statement

Our studies did not include human participants, human data or human tissue. All the animal protocols containing all the procedures were approved by the ethics committee of Beijing Neurosurgical Institute Ethics Committee of Capital Medical University. The experimental protocols were conducted in accordance with the ARRIVE guidelines.

Conflict of interest

The authors declare no competing interests.

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You are reading this latest preprint version

Extracorporeal shock waves effectively suppressed the proliferation and growth of colorectal cancer

Status:

Version 1

Abstract

Figures

1. Introduction

2. Results

2.1 Impact of stepwise increased dosage of ESV energy on CRC xenograft tumor growth

2.2 Administration of 60 mJ ESV delayed the growth of CRC-derived xenografts

2.3 Administration of 60 mJ ESV blocks CRC cell proliferation

2.4 ESV induces apoptosis and ferroptosis in CRC cells

2.5 Effect of ESV on cell proliferation and death is related to the p53 pathway

2.6 ESV inhibits proliferation and promotes death of CRC cells in vitro

3. Discussion

4. Materials and Methods

4.1 Mouse Xenograft Studies

4.2 ESV application and definition

4.3 RNA Sequencing

4.4 Immunohistochemistry (IHC) analysis

4.5 Reverse Transcription-Quantitative Polymerase Chain Reaction

4.6 Western blotting

4.7 Statistical analysis

Declarations

Funding

Contributions

Data availability

Competing interests statement

Ethic statement

Conflict of interest

References

Additional Declarations

Supplementary Files

Status:

Version 1