Lidocaine could promote the cuproptosis through up-regulating the long noncoding RNA DNMBP-AS1 in laryngeal cancer

doi:10.21203/rs.3.rs-2956270/v3

Download PDF

Research Article

Lidocaine could promote the cuproptosis through up-regulating the long noncoding RNA DNMBP-AS1 in laryngeal cancer

https://doi.org/10.21203/rs.3.rs-2956270/v3

This work is licensed under a CC BY 4.0 License

Version 3

posted

You are reading this latest preprint version

Background: Lidocaine is a traditional local anesthetic, which has been reported to trigger apoptosis through the mitochondrial pathway, independent of death receptor signaling. Cuproptosis is a copper triggered mitochondrial cell death mode. In this study, we explored the biological effects of lidocaine on laryngeal cancer and studied the relevant mechanisms of cuproptosis

Methods: quantitative RT-PCR weas used to measure the expression level of long noncoding RNA (IncRNA) DNMBP-AS1. DNMBP-AS1 siRNA (si-DNMBP-AS1) were transfected into Hep-2 cells to verify the roles of DNMBP-AS1 in cuproptosis. 24 hours treatment with 20 nM elesclomol and 2 µM CuCl2 was performed to promote the occurrence of Cuproptosis. Cell proliferation and apoptosis assays ware utilized to analyze biological effect of lidocaine on Hep-2 cells.

Results: DNMBP-AS1 was significantly upregulated during cuproptosis in Hep-2 cells. The si-DNMBP-AS1 significantly increased the cell viability, and suppress the cuproptosis. Lidocaine was cytotoxic to the Hep-2 cells in a dose- and time-dependent manner. Exposure to 10 μM of lidocaine for 24 hours did not reduce the viability, but significantly increased the expression of DNMBP-AS1, and promote the cuproptosis. Anymore, si-DNMBP-AS1 reverse the pro-cuproptosis function of lidocaine.

Conclusions: lidocaine was cytotoxic to human laryngeal cancer cells in a time- and dose-dependent manner, promoted the cuproptosis through up-regulating DNMBP-AS1. The results of this study offer initial optimism that lidocaine can be used in an adjuvant or neoadjuvant fashion in laryngeal cancer treatment.

lidocaine

laryngeal cancer

cuproptosis

IncRNA DNMBP-AS1

Laryngeal cancer is a common cancer with high incidence rate and mortality. In countries with advanced health care systems, the mortality rate of laryngeal cancer is decreasing, but the incidence rate in developing countries is still high, and this disease is the second most common head and neck cancer in the world [1, 2]. Therefore, it is important to further explore the molecular mechanism of laryngeal cancer and find new therapeutic targets to improve the prognosis.

Cuproptosis is a new cell death mode, which is related to copper and mitochondrial respiration [3]. The pathological mechanism is that copper directly interacts with the fatty acylated components of the tricarboxylic acid (TCA) cycle, resulting in excessive aggregation of fatty acylated proteins and loss of iron sulfur cluster proteins, thereby stimulating proteotoxic stress and cell death. Recent studies have found that copper cell death is closely related to human cancer [4-6], proving that copper ptosis is closely related to the occurrence and development of cancer, but it is not clear in laryngeal cancer.

Long non-coding RNAs (LncRNAs) play a key role in gene regulation because they can affect cell proliferation, migration and genome stability. In recent years, transcriptome sequencing has revealed that thousands of aberrantly expressed lncrnas are associated with different cancers [7-9]. The mechanisms of action of lncrnas in carcinogenesis include enhancing chromatin state and methylation, maintaining the stability of proteins or protein complexes, and acting as sponges for inhibiting microRNAs (miRNAs). Previous studies have shown that lncRNA DNMBP-AS1 is significantly associated with poor overall survival in patients with breast cancer and colon cancer [10, 11]. A recent study reported that DNMBP-AS1 may inhibit the progression of hepatocellular carcinoma through cuproptosis induction [12].

Lidocaine is a kind of amide local anesthetic. It has been found that lidocaine has good potential in anti-tumor treatment [13, 14]. However, the role of lidocaine in laryngeal cancer remains unknown. A previous study demonstrated that lidocaine induces apoptosis through the mitochondrial pathway, independent of death receptor signaling [15].

In this study, we explored the biological effect of lidocaine on laryngeal cancer and the related mechanism of cuproptosis, which is expected to provide more theoretical references for the treatment of laryngeal cancer.

cell culture and treatment

The Hep-2 cell lines were purchased from ATCC (American Type Culture Collection). The cells were cultyred in Dulbecco's modified Eagle's medium (DMEM; Gibco Corporation, USA) with 10% fetal bovine serum (HyClone, USA) and 1% penicillin/streptomycin (Invitrogen) at 37°C with 5% CO₂. To evaluate the effect of lidocaine, we seeded the Hep-2 cells at a density of 4000 cells/well in 96-well plates in triplicate. Once they reached 80% confluence, the cells were treated for 24 hours, 48 hours and 72 hours with various concentrations of lidocaine (1 μM, 5 μM, 10 μM, 25 μM, 50 μM and 100 μM) and compared with a control. For cuproptosis occurrence, cells were treated with 20 nM elesclomol and 2 µM CuCl2 for 4 hours, 12 hours and 24 hours.

Cell transfection

si-DNMBP-AS1 and negative control (si-ctrl) were synthesized by GenePharma (Shanghai, China). Hep-2 ccells were uniformly plated in 96 well plates. When the cells reached about 80-90% fusion, they were transfected with plasmid by Lipo3000 (Invitrogen, Carlsbad, CA, USA). Cells were harvested for the subsequent experiments following incubation at 37°C for 48 h.

The quantitative real-time polymerase chain reaction (qRT−PCR)

Trizol (Beyotime, Shanghai, China) was used to extract the total RNA from LUAD cells and tissues. RNA was reverse-transcribed into complementary DNA (cDNA) using SuperScript VILO cDNA Kit (Thermo Fisher Scientific, Inc.). The primer sequences for PCR were as follows: DNMBP-AS1 forward, 5′-TCGCTCCTCATAGCGAGTCT-3′ and reverse, 5′-TGACTTCCTTATCCGCTCCC-3′; and GAPDH forward, 5′-GGAGCGAGATCCCTCCAAAAT-3′ and reverse, 5′-GGCTGTTGTCATACTTCTCATGG-3′. The real-time PCR was performed with a SYBR Green PCR kit (Takara Biotechnology Co., Ltd., Dalian, China) to measure the expression of DNMBP-AS1 and GAPDH. The data were analyzed according to the 2^−△△CTmethod .

Cell proliferation assay

96 well plates were used to seed Hep-2 cells (2 × 10⁵ cells / well). After incubation at 37 ° C and 5% CO₂ for different times, the CCK-8 kit provided by Tiangen (Hangzhou, China) was mixed at 10 µ L / well, and cells were incubated for 3 hours at 37 ° C and 5% CO₂. Finally, we read the absorbance at 450 nm on the microplate reader (Thermo Fisher Scientific, Inc.)

Cell death analysis

Cells were seeded in six-well plates at the density of 1 × 10⁵ cells/well. Each well contained 2 ml of culture medium.Cells were incubated for 24 hours and then were exposed to the anti-androgen media containing C-S serum and Bicalutamide for 24 h. Cells then were collected and rinsed with PBS and re-suspended in 300 μl of binding buffer. Next, 5 μl of PI was added to the cell suspensions and incubated for 15 min in the dark. Cell death was then detected by using a BD FACSVerse flow cytometer.

Statistical analysis

One-way analysis of variance (ANOVA) were used to assess differences between groups. SPSS 25.0 software was performed for statistical analyses. All experiments were performed independently and repeated three times. P < 0.05 was considered statistically significant.

DNMBP-AS1 was significantly upregulated by exogenous introduction of copper ions

We first evaluated whether copper could affect the expression of the DNMBP-AS1. Elesclomol is a copper ionophore that shuttles copper into the cell. We treated laryngocarcinoma cell lines with copper chloride (2μM) combined with elesclomol (20nM) for 24h. The results of qRT-PCR showed that DNMBP-AS1 was significantly upregulated during cuproptosis (Figures 1A). Then, CCK-8 assay showed that si-DNMBP-AS1 significantly increased the cell viability of Hep-2 cells (Figure 1B,C). Cell death analysis showed that si-DNMBP-AS1 could significantly suppress the cuproptosis (Figure 1D). The results confirm that DNMBP-AS1 plays a important role in cuproptosis in laryngocarcinoma.

Lidocaine could promote the cuproptosis through up-regulating the DNMBP-AS1

CCK-8 assay showed that lidocaine was cytotoxic to the laryngocarcinoma cell line in a dose- and time-dependent manner (Figure 2A). Interestingly, exposure to 10 μM of lidocaine for 24 hours did not reduce the viability of Hep-2 cells, but significantly increased the expression of DNMBP-AS1 (Figure 2B). Cell death analysis showed that exposure to 10 μM of lidocaine for 24 hours could significantly promote the cuproptosis in Hep-2 cells. Anymore, si-DNMBP-AS1 reverse the pro-cuproptosis function of lidocaine (Figure 2C). The results indicated that lidocaine could promote the cuproptosis through up-regulating the DNMBP-AS1 in laryngocarcinoma.

The concept cuproptosis was was proposed in March 2022. So far, there are limited reports on this issue in the literature [16, 17]. The in-depth study of cuproptosis is helpful to promote the new understanding of tumor pathogenesis. The function and regulatory role of lncRNAs in cuproptosis are increasingly recognized. Xie et al. constructed a risk model based on cuproptosis-related lncRNAs can accurately predict the prognosis of cutaneous melanoma [18]. Liu et al. identified eight cuprotosis-related lncRNAs signature of HNSC as promising biomarkers for predicting the benefit of head and neck squamous cell carcinoma from immunotherapy [19]. Wang et al. constructed a ten-cuproptosis-related lncRNA signature that provided promising insights into personalized prognosis and drug selection among colon adenocarcinoma patients [17]. Xu et al. reported that cuproptosis-related lncRNA MIR31HG could act as an oncogene in lung adenocarcinoma [20]. However, no studies to date have assessed the role of cuproptosis-related lncRNAs in laryngocarcinoma. A recent study reported that DNMBP-AS1 could partake in the cuproptosis induction [12]. In this study, we found that expore to copper chloride combined with elesclomol significantly upregulated the expression of DNMBP-AS1 in Hep-2 cells. The si-DNMBP-AS1 significantly increased the cell viability, and suppressed the cuproptosis, which indicated that DNMBP-AS1 could play a important role in cuproptosis in laryngocarcinoma.

Previous studies have shown that local anesthetics are toxic to multiple cancer types. Tu et al. Reported that lidocaine could prevent breast cancer growth by targeting neuronatin to inhibit nerve fibers formation [21]. Xu et al. found that lidocaine could enhance the effects of chemotherapeutic drugs against bladder cancer [13]. Wan et al. showed that lidocaine could impede the tumor progression in hepatocellular carcinoma via circ-DYNC1H1/miR-520a-3p/USP14 axis. [22]. However, the biological effects of lidocaine on laryngocarcinoma remains to be researched. In this study, we found that lidocaine had slight cytotoxic effect on laryngocarcinoma cell line in a dose- and time-dependent manner. Though exposure to 10 μM of lidocaine for 24 hours did not reduce the viability of Hep-2 cells, it significantly increased the expression of DNMBP-AS1, and enhanced the cuproptosis in Hep-2 cells, which was reversed by si-DNMBP-AS1. These results indicated that lidocaine could promote the cuproptosis through up-regulating the DNMBP-AS1 in laryngocarcinoma. Stevens et al. found that death receptors were not involved in lidocaine-induced cell death, which could be mediated by the intrinsic mitochondrial death pathway [15]. Interestingly, cuproptosis has also been demonstrated as a copper dependent regulated form of cell death driven by aggregation of mitochondrial lipoacylated proteins [16,17].

This study found that lidocaine was cytotoxic to human laryngocarcinoma cells in a time- and dose-dependent manner, promoted the cuproptosis through up-regulating DNMBP-AS1. The results of this study are initially optimistic that lidocaine can be used as an adjuvant or neoadjuvant modality in the treatment of laryngeal cancer, and further studies should be conducted to determine whether these results can be translated into a clinical environment.

Disclosure

-Conflict of interest : The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

-Ethics approval: This study did not involve human participants or human participants’ data.

-Registry and the Registration No. of the study/trial :N/A

-Informed Consent: N/A

-Animal Studies : N/A.

Funding

This study was supported by the Basic scientific research project of Liaoning Provincial Education Department [LJKZ0858].

Financial Disclosure statement

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Authors' contributions

Y Yu. : Methodology, Software, Data Curation, Writing - Original Draft.

Y He: Methodology, Software, Data Curation, Writing - Original Draft.

MH Lv: Methodology, Writing - Review & Editing, Supervision, Project administration.

Availability of data and materials

All data generated or analysed during this study are included in this published articl

[1] Zhang Z-M, Zhao Z, Chen Z-X. Impact of Laryngocarcinoma at Different Sites in 16,255 Individuals. Ear, Nose & Throat Journal. August 2021.

[2] Su, J., Lu, E., Lu, L. and Zhang, C. (2017), MiR-29a-3p suppresses cell proliferation in laryngocarcinoma by targeting prominin 1. FEBS Open Bio, 7: 645-651.

[3] Wang, Y., Zhang, L. & Zhou, F. Cuproptosis: a new form of programmed cell death. Cell Mol Immunol 19, 867–868 (2022).

[4] Hu Q, Wang R, Ma H, Zhang Z and Xue Q (2022) Cuproptosis predicts the risk and clinical outcomes of lung adenocarcinoma. Front. Oncol. 12:922332.

[5] Song Q, Zhou R, Shu F and Fu W (2022) Cuproptosis scoring system to predict the clinical outcome and immune response in bladder cancer. Front. Immunol. 13:958368.

[6] Li, L., Li, L. & Sun, Q. High expression of cuproptosis-related SLC31A1 gene in relation to unfavorable outcome and deregulated immune cell infiltration in breast cancer: an analysis based on public databases. BMC Bioinformatics 23, 350 (2022).

[7] Zhan, L., Li, J. & Wei, B. Long non-coding RNAs in ovarian cancer. J Exp Clin Cancer Res 37, 120 (2018).

[8] Rao, A.K.D.M., Rajkumar, T. & Mani, S. Perspectives of long non-coding RNAs in cancer. Mol Biol Rep 44, 203–218 (2017).

[9] Huang, Y, Jiang, L, Liu, Y, Liu, L, Wang, J, Shi, L. Long non-coding RNAs in virus-related cancers. Rev Med Virol. 2022;e2364.

[10] Gao S, Lu X, Ma J, Zhou Q, Tang R, Fu Z, Wang F, Lv M and Lu C (2021) Comprehensive Analysis of lncRNA and miRNA Regulatory Network Reveals Potential Prognostic Non-coding RNA Involved in Breast Cancer Progression. Front. Genet. 12:621809.

[11] Yang L, Yang T, Wang H, Dou T, Fang X, Shi L, Li X and Feng M (2022) DNMBP-AS1 Regulates NHLRC3 Expression by Sponging miR-93-5p/17-5p to Inhibit Colon Cancer Progression. Front. Oncol. 12:765163.

[12] Zhang, G., Sun, J. & Zhang, X. A novel Cuproptosis-related LncRNA signature to predict prognosis in hepatocellular carcinoma. Sci Rep. 2022; 12, 11325.

[13] Zhong Wang, Qin Liu, Jun Lu, Jian Cao, Xiao-Yan Wang, Yong Chen. Lidocaine promotes autophagy of SH-SY5Y cells through inhibiting PI3K/AKT/mTOR pathway by upregulating miR-145. Toxicology Research. 2020; 9: 467–473.

[14] Zhang C, Xie C and Lu Y (2021) Local Anesthetic Lidocaine and Cancer: Insight Into Tumor Progression and Recurrence. Front. Oncol. 11:669746.

[15] Robert Werdehausen, Sebastian Braun, Frank Essmann, Klaus Schulze-Osthoff, Henning Walczak, Peter Lipfert, Markus F. Stevens; Lidocaine Induces Apoptosis via the Mitochondrial Pathway Independently of Death Receptor Signaling. Anesthesiology 2007; 107:136–143

[16] Tang, D., Chen, X. & Kroemer, G. Cuproptosis: a copper-triggered modality of mitochondrial cell death. Cell Res 32, 417–418 (2022).

[17] Xu M, Mu J, Wang J, Zhou Q and Wang J (2022) Construction and validation of a cuproptosis-related lncRNA signature as a novel and robust prognostic model for colon adenocarcinoma. Front. Oncol. 12:961213.

[18] Zhou Y, Shu Q, Fu Z, Wang C, Gu J, Li J, Chen Y and Xie M (2022) A novel risk model based on cuproptosis-related lncRNAs predicted prognosis and indicated immune microenvironment landscape of patients with cutaneous melanoma. Front. Genet. 13:959456.

[19] Yang L, Yu J, Tao L, Huang H, Gao Y, Yao J and Liu Z (2022) Cuproptosis-Related lncRNAs are Biomarkers of Prognosis and Immune Microenvironment in Head and Neck Squamous Cell Carcinoma. Front. Genet. 13:947551.

[20] Mo X, Hu D, Yang P, Li Y, Bashir S, Nai A, Ma F, Jia G and Xu M (2022) A novel cuproptosis-related prognostic lncRNA signature and lncRNA MIR31HG/miR-193a-3p/TNFRSF21 regulatory axis in lung adenocarcinoma. Front. Oncol. 12:927706.

[21] Bingda Li, Hao Xu, Chongwu He, Wenxiong Zou, Yun Tu. Lidocaine prevents breast cancer growth by targeting neuronatin to inhibit nerve fibers formation.The Journal of Toxicological Sciences 2021 ; 46 :329-339.

[22] Liu, Hua, Cheng, Jing, Xu, Heng and Wan, Zhenzhen. "Lidocaine has antitumor effect on hepatocellular carcinoma via the circ_DYNC1H1/miR-520a-3p/USP14 axis" Open Life Sciences, vol. 16, no. 1, 2021, pp. 766-780.

No competing interests reported.

Download PDF

Version 3

posted

You are reading this latest preprint version

Lidocaine could promote the cuproptosis through up-regulating the long noncoding RNA DNMBP-AS1 in laryngeal cancer

Status:

Version 3

Abstract

Figures

Background

Methods

Results

Discussion

Conclusions

Declarations

References

Additional Declarations

Status:

Version 3