Bee venom induced the interaction of phosphorylated histone variant, γH2AX, and intracellular location of beta actin in cancer cells

doi:10.21203/rs.3.rs-1528971/v1

Download PDF

Research Article

Bee venom induced the interaction of phosphorylated histone variant, γH2AX, and intracellular location of beta actin in cancer cells

https://doi.org/10.21203/rs.3.rs-1528971/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Bee venom is a natural compound and candidate anti-cancer agent with selective cytotoxic effect on some cancer cells. However, the cellular mechanisms of how bee venom selectively targets cancer cells remain elusive. The aim of this study was to reveal the genotoxic effect of bee venom in concordance with the location of β-actin protein throughout nucleus or/and cytoplasm. For this aim, the level of H2AX phosphorylation (γH2AX) and intracellular location of β-actin were assessed by immunofluorescence in liver (HEPG2) and metastatic breast (MDA-MB-231) cancer cell lines compared to normal fibroblasts (NIH3T3) after bee venom. Co-localisation of γH2AX and β-actin were also analysed. The results showed that the levels of γH2AX staining decreased in normal cells but increased in cancer cells. Majority of β-actin was localised within the cytoplasm of normal cells after bee venom, but it was mostly accumulated within the nucleus in cancer cells. Co-localisation of β-actin and γH2AX both in nucleus and cytoplasm was induced in each cell by different patterns. The results showed that normal and cancerous cells had different responses against bee venom, and suggested that bee venom induced a cellular response by the interaction between γH2AX and β-actin.

Bee venom

cancer

histone variant

H2AX

actin

DNA damage

genotoxicity

Alternative medicine, also called complementary medicine, is defined as the use of natural plant- or animal-derived products for the treatment of various diseases. Apitherapy is one of the alternative medicine approaches that uses bee products e.g. bee venom (BV) for therapeutic purposes. A range of in vitro and in vivo studies have revealed that BV has analgesic, anti-inflammatory, anti-microbial, anti-viral and anti-cancer activities (Son et al., 2007; Uddin et al., 2016). Its anti-cancer activity has been shown in various cancer cells including prostate, breast, lung, liver, skin and bladder cancers by induction of cell death and growth arrest (Orsolic, 2012; Tu, Wu, Hsieh, Chen, & Hsu, 2008). BV is composed of a range of enzymes, peptides and amines including melittin and phospholipase A2, apamin, histamine, dopamine, norepinephrine, adolapine, and mast cell-degrading peptides (Huh et al., 2010; Lariviere & Melzack, 1996; Son et al., 2007). These components of bee venom can affect cancer cells with different cellular pathways. For example, it has been shown that bee venom induced cell death in glioblastoma cells by suppressing the effect of matrix metalloprotease-2 enzyme (Sisakht, 2017). In addition, phospholipase A2 found in bee venom (Doery & Pearson, 1964; Putz et al., 2007; Sobotka et al., 1976) selectively acts on cancer cells with damaged membranes, but cannot penetrate normal cells with intact membranes (Putz et al., 2007). Melittin, the major component of bee venom and a lytic peptide, can integrate to the hydrophobic layer of the membrane and target the membrane (Leuschner & Hansel, 2004). BV inhibited the proliferation of cancer cells by stimulating the cellular immune response in the lymph nodes (Liu, Chen, Xie, & Zhang, 2002; Orsolic et al., 2003). While bee venom induced apoptosis in human leukemia cells; it did not cause cytotoxicity in healthy bone marrow cells through some mechanisms (Hong et al., 2005; Jang et al., 2003; Moon et al., 2006) suggesting that bee venom is a promising agent for selective cytotoxicity on cancer cells.

Cancer is a disease defined by uncontrolled cell proliferation, and cell invasion to other tissues while normal cells maintain their orginal locations and the balance between cell proliferation and cell death by strict cellular signals. Although the main focus of cancer treatments is to selectively induce cell death in malignant cells, many of current chemotherapeutics are the lack of cytotoxic selectivity for cancer cells. A part of investigations have targeted to find natural or synthetic new therapeutics that have non-cytotoxic effects on normal cells but cytotoxic effects on cancer cells (Tu et al., 2008; Zheng et al., 2015), and bee venom can be one of the candidate for this purpose (Hong et al., 2005; Ip et al., 2008; Jang et al., 2003; Jeong et al., 2014; Jo et al., 2012). Although the cytotoxic effect of bee venom has been shown in various cancer cells (Moga, Dimienescu, Arvatescu, Ifteni, & Ples, 2018), the mechanisms of its selectivity for cancer cells remain unclear. In our previous study, the selective effect of bee venom on cancer cells has been confirmed in metastatic breast cancer and hepatocellular cancer cell lines and the study also showed that cells undergo different patterns of epigenetic reprogramming after bee venom (Uzuner, Birinci, Tetikoglu, Birinci, & Kolayli, 2021). However, to the best of knowledge, there is no understanding on the cellular mechanisms of bee venom for this selectivity in terms of inducing DNA damage by double strand breaks (DSBs) and altering cytoskeleton localisation, in cancer cells compared to normal cells.

This study aims to reveal the response of cells against bee venom in terms of inducing DNA damage and repair by H2AX phosphorylation and β-actin location within the cells. The findings suggest that bee venom induces DNA damage mediated by H2AX phosphorylation and intra-cellular mobility of β-actin in cancer cells compared to normal cells.

2.1 Bee venom collection and preparation

Bee venom used was collected as stated previously and SDS-page analyses were also done (Uzuner et al., 2021). Bee venom main stock was prepared in 0.9% NaCl and stocked as 5mg/ml aliquots at -20°C.

2.2 Cell culture

Cells used were MDA-MB-231 human metastatic breast cancer cells (American Type Cell Collection ATCC, Cat No. HTB-26, VA, USA), HEPG2 human liver cancer cells (ATCC, Cat. No. HB-8065), and NIH3T3 embryonic mouse fibroblast (ATCC, Cat. No. CRL-1658) cells. MDA-MB-231, HEPG2 and NIH3T3 cells were cultured in RPMI (Wisent Inc. Multicell, USA, Cat. No. 350000CL), EMEM (Cat. No. 320026CL) and DMEM (Cat. No. 319005CL) media, respectively. These media contained 10% (v/v) fetal bovine serum (Sigma-Aldrich Co. St. Louis, US, Cat. No: 12103C) for MDA-MB-231 and HEPG2 cells and 10% (v/v) bovine calf serum (Sigma-Aldrich, Cat. No. 12133C) for NIH3T3 cells, and 1% (v/v) penicillin-streptomycin (Capricorn, Cat. No. PS-B) was added to each media. Cultures were incubated at 37 °C incubator with 5% CO2.

2.3 Immonuflorescence for DNA Damage (γH2AX) and β-actin

MDA-MB-231, HEPG2 and NIH3T3 cells were cultured in 96 well plates at 5000 cells per well and incubated overnight (O/N). After treatment with bee venom at concentrations of 0, 8, 12, 25, and 50 μg/mL for 24 h, cells were washed and fixed with 4% (w/v) paraformaldehyde (PFA) (ChemSolute 135 Bio, Cat No. 8416. 0500, Germany) for 30 min at room temperature (RT). Cells were treated with 1x PBS containing 0.5% Triton-100 (Biomatik, Cat No. A4025, ON, Canada) for 15 min at RT to permeabilize the cells. Subsequently, cells were blocked with 1x PBS containing 20% (v/v) goat serum (Capricorn, Cat. No. GOA-1B), 200 mg/mL BSA (Sigma, Cat. No. A1470) and 0.01% (v/v) Tween-20 (Sigma, Cat No. P1379) for 2 h at 37°C. Cells were then treated with mouse polyclonal primary antibody ƴ-H2AX (1:200) (Aviva systems biology, Cat. No. OAAN02886) in 1x PBS containing 20 mg/mL BSA and 0.05% triton-100 (v/v) at 4°C overnight (O/N) Control cells were incubated only with secondary antibodies. After incubation, the primary antibody was washed 3 times with 1x PBS then was treated with secondary Goat Anti-Mouse IgG H&L (1:1000) (Texas Red®) (ab6787) antibody in 1x PBS including 20 mg/mL BSA and 0.05% triton-100 (w/v) at 4°C O/N. After incubation it was washed with 1x PBS three times. For β-actin staining, cells were differently treated with the mouse monoclonal primary antibody anti-β-actin (1:250) (BioLegend, Cat. No. 643801) prepared in 1x PBS containing 20 mg/mL BSA and 0.05% triton-100 (w/v) for 1h in the dark followed by the washing with 1x PBS three times. Cells were then treated with secondary antibody (1:1000) Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) (ab150113) prepared in 1x PBS containing 20 mg/mL BSA and 0.05% triton-100 (v/v) for 1 hour in the dark at RT. Secondary antibody was removed by washing with 1x PBS three times. As the last step, cells were incubated with 4 μg/mL bis Benzimide Hoechst 33342 (Sigma, Cat No. 14533) prepared in 1x PBS to label the DNA. Finally, each well was examined under the inverted fluorescence microscope (AxioVert A1, Zeiss, Germany), and images were taken as at least five different regions per treatment. ImageJ software (NIH, USA) was used to analyse the level of staining for γH2AX. The intra-cellular localisation of β-actin was analysed by grouping cells according to staining pattern as i) in cytoplasm only, ii) in nucleus only, and iii) in both cytoplasm and nucleus, and the cells for each group were counted.

2.4. Statistical Analyses

Statistical analyses were performed with SPSS software (Version 23). The staining level of γH2AX and the number of cells with different β-actin localisation were compared using UNIANOVA (univariate analysis of variance). The Post-Hoc tests of UNIANOVA were performed for pairwise comparisons. p<0.05 (*), p<0.01 (**), p<0.001 (***) and p<0.0001 (****) were used for significance values in comparisons.

3.1 Detection of DNA Damage

Immunostaining was performed for γH2AX, a DNA damage marker, after 24 hours of BV treatment to understand whether bee venom has a genotoxic potential on NIH3T3, MDA-MB-231 and HEPG2 cells (Fig. 1). The levels of γH2AX staining decreased in normal NIH3T3 cells (Fig. 1A-1D), but increased in HEPG2 (Fig. 1B-1E) and MDA-MB-231 (Fig. 1C-1F) cells after bee venom. HEPG2 and MDA-MB-231 cells were highly sensitive to BV at the highest dose (50 µg/mL) (Fig. 1E-1F). The previous study showed that IC50 values for NIH3T3, MDA-MB-231 and HEPG2 cells were 50 µg/mL, 8 µg/mL and 12 µg/mL, respectively (Uzuner et al., 2021).

3.2 Detection of β-actin

β-actin immunostaining was performed to determine its cellular distribution in normal and cancer cells after bee venom treatment (Fig. 2). In normal cells, majority of β-actin was localised within the nuclei of NIH3T3 cells, but it was distributed along with the cytoplasm in a dose-dependent manner after BV (Fig. 2A-2D). In contrast to normal cells, the β-actin was found both in the cytoplasm and nucleus in untreated HEPG2 cancer cells, while almost all were localized within the nucleus at 25 and 50 µg/mL doses of bee venom (Fig. 2B-2E). The β-actin that generally distributed within the cytoplasm in MDA-MB-231 cancer cells, but it was localized more in the nuclei at 25 and 50 µg/mL suggesting that both cancer cell lines responded against cytotoxic doses of bee venom in a similar way with an increase in nuclear accumulation of β-actin (Fig. 2C-2F). The major intracellular location of β-actin is given in Table 1.

3.3. Co-localisation of β-actin and γH2AX

To understand the interaction between β-actin and γH2AX after bee venom treatment, cells were co-stained for both β-actin and γH2AX, and merged profiles were obtained. In MDA-MB-231 cells, β-actin and γH2AX were not co-localised within the cells as β-actin was distributed thoughout the cytoplasm (blue arrow) but γH2AX was located within the nuclei (white arrow) in normal cells (Fig 3). Bee venom treatment resulted in increased co-localisation of two markers within both cytoplasm and nucleus. In HEPG2 cells, β-actin and γH2AX were mostly found co-localised in both untreated and bee venom-treated cells (white and red arrows for nuclear and cytoplasmic colocalisation, respectively) (Fig 4). But in untreated cells, β-actin staining was not co-localised with γH2AX to some extent (blue arrows). With bee venom, co-localisation profiles both in nucleus and cytoplasm were more distinctively observed. In NIH3T3 cells, bee venom induced some cytoplasmic co-existence of β-actin and γH2AX (blue arrows), however nuclear co-localisation also occurred (white arrows) (Fig 4). Table 2 summarises the co-localisation pattern of β-actin and γH2AX in each cells.

Bee venom treatment induced β-actin to localise throughout the nuclei of cancer cells in accordance with an increase in phosphorylation of H2AX, but significantly found in the cytoplasmic form in non-cancerous cells in accordance with a decrease in γH2AX nuclear accumulation. The histone H2AX used is a 14 kDa member of the H2A histone family, which is evolutionarily conserved at the C-terminus in eukaryotes. Serine 139 within this motif is rapidly phosphorylated in response to double-stranded DNA damage and apoptosis, and its phosphorylated form is known as γH2AX. Phosphorylation reaches its maximum level at 1–3 minutes after DNA damage occurs, and hundreds of γH2AX molecules are released during each double-strand DNA break (DSBs) (Schmid, Zlobinskaya, & Multhoff, 2012) and the H2AX phosphorylation is the most fundamental modification involved in DNA damage (Sedelnikova, Pilch, Redon, & Bonner, 2003; Sonoda, Hochegger, Saberi, Taniguchi, & Takeda, 2006; Stucki et al., 2005). Therefore the immunostaining pattern of γH2AX has been used for a marker of DSBs and genomic instability in cancer cells (Hamer et al., 2003; Ji et al., 2017; Nagelkerke & Span, 2016; Redon, Dickey, Bonner, & Sedelnikova, 2009). But, H2AX phosphorylation has been also observed in undamaged cells in human (Meyer et al., 2013). To determine whether bee venom has a genotoxic effect on NIH3T3, MDA-MB-231 and HEPG2 cells, immunostaining was performed for γH2AX after bee venom treatment for 24 h, and γH2AX staining was decreased in normal cells while increased in cancer cells after bee venom. The level of DSBs increases with cellular death, chromosomal aberrations, mutations and initiation of pathological effects such as cancer so that repairing of DSBs is critical and essential to prevent carcinogenesis (Kasparek & Humphrey, 2011). Histone modifications associated with DNA damage allow DNA repair factors to access damaged sites of DNA (Waterman, Haber, & Smolka, 2020). H2AX is mostly found in nuclues, but It has been previously reported that phosphorylated H2AX was also accumulated within the cytoplasm in some DNA damage induced by such overactivation of a nerve growth receptor (Jung & Kim, 2010, 2011). In each cells some of γH2AX protein were found in cytoplasm after bee venom. But, the cellular pathway for γH2AX protein accumulation after bee venom needs further investigation.

Cytoskeleton is structured by various proteins such as microtubules, actin and intermediate filaments (Fletcher & Mullins, 2010). The cytoskeletal proteins have important roles at cell movement, biochemical process of cell, and survival of the cell (Ong et al., 2020). Cancer cells have altered skeletal machinery that causes cell invasion and metastasis (Pawlak & Helfman, 2001). Actins have three isoforms including α-, β- (Β-actin), and γ-actin and also they are involved in forming microfilaments in the cell (Kavallaris, 2010; Rao & Li, 2004). While normal cells strictly establish cell architecture, mobility and adhesion with actin crosslinks, cancer cells disrupt the actin cytoskeleton arrangement and change the nuclear:cytoplasmic ratio in cells. This facilitates the metastasis by inducing flexible, mobile and fast movement of cancer cells (Ong et al., 2020; Rao & Li, 2004; Tojkander, Gateva, & Lappalainen, 2012).

Defects in cellular morphogenesis are accompanied by uncontrolled migration, acquisition of invasive features, and genomic instability in carcinogenesis. Cancer cells have abnormalities in each cellular compartment as in nucleus, cytoplasm and membranes. Cytoplasmic rearrangement mediate cell mobility and its abnormalities are one of the key events in cancer cells. For example, actin and tubulin proteins involve in the regulation of intracellular compartments, cell polarity and contractility. During morphogenesis, they determine cell shape and polarity and also promote stable cell-cell and cell-matrix junctions through interactions with cadherin and integrin proteins, respectively (Eden, Rohatgi, Podtelejnikov, Mann, & Kirschner, 2002). Additionally they activate chromosomal segregation and cell division during mitosis. This study showed that β-actin protein is localized both in the cytoplasm and in the nucleus depending on the bee venom response in different cells. The findings support previous studies that have shown the localisation of β-actin within both cytoplasm and nucleus (Caridi et al., 2018; Cerutti, 2019; Schrank et al., 2018). β-actin in the cytoplasm is involved in cell movement, formation of the skeleton and cell division, while β-actin in the nucleus is involved in the G2 checkpoint where especially the repair of DSBs occurs (Schrank et al., 2018). In addition, it has been determined that β-actin has various structural and functional roles in the nuclear structure, such as nuclear matrix assembly, chromatin remodeling, transcription and mRNA processing (Bohnsack, Stuven, Kuhn, Cordes, & Gorlich, 2006). Mass spectrometry (Bohnsack et al., 2006) and immunoreactivity experiments (Hu, Wu, & Hernandez, 2004) have showed that β-actin is a nuclear isoform associated with heterogeneous nuclear ribonucleoproteins (hnRNPs) and RNA polymerase complexes. It has been also shown that different polymerization states of β-actin coexist in the nucleus (Schoenenberger et al., 2005). Fluorescent staining studies have revealed that approximately 20% of the nuclear actin has a dynamic pattern (McDonald, Carrero, Andrin, de Vries, & Hendzel, 2006). Our results showed that bee venom differentially induced the re-organisation of cytoskeleton but similarly induced DNA damage suggesting that bee venom selectively can stimulate β-actin-mediated DNA repair within the nucleus in cancer cells. Nuclear form of β-actin has been recently shown to mediate re-organisation of nucleolus after DNA repair particularly in ribosomal DNA sequences which is completed after UV-induced damage (Caridi et al., 2018). Another study showed that γH2AX phosphorylation increased in cells after radiation, followed by the binding of NMI (nuclear myosin 1) to ribosomal DNA (rDNA), and subsequent β-actin molecules were recruited into damaged cell nuclei (Cerutti, 2019) after phosphorylated H2AX-mediated binding of NMI to the damaged site (Caridi et al., 2018). NMI and β-actin are required for appropriate rearrangement of the nucleolus after the completion of the DNA repair machinary. Results showed that genotoxicity increased after bee venom in cancer cells but not in normal cells, and β-actin localised within the nuclei in cancer cells. This suggests that β-actin may recruit to damaged site within the nucleus in coordination with γH2AX in the cell nucleus to manage DNA damage. The results in this study indicate that after bee venom treatment, β-actin was accumulated more in the nuclei of cancer cells and but in the cytoplasm of normal cells. Thus, it suggests that bee venom may act by interfering with chromatin changes and mRNA processing of β-actin in cancer cells.

This study attempted to understand i) the genotoxic effect of bee venom in cancer cells by a histone variant, H2AX, phosphorylation and ii) the association of β-actin with this genotoxicity. Results showed that normal and cancerous cells had different responses against bee venom, and suggested that bee venom induced a cellular response by the interaction of γH2AX and β-actin. Overall, this study revealed one of cellular mechanism in cancer cells after bee venom. The results add detailed knowledge for consideration of the use of bee venom as alternative medicine in therapies of some cancers.

Acknowledgements

This study was supported by grants (FYL-2019-8116 and FBA-2018-7951) from Scientific Research Projects Coordination Unit of Karadeniz Technical University. Authors thank to Prof Sevgi Kolaylı (Department of Chemistry, Karadeniz Technical University) for bee venom supplies, and Mrs Esra Birinci (Department of Biochemistry in Pharmacology, Karadeniz Technical University) for performing previous MTT cytotoxicity assays. And the best outcome of this study was to marry Mrs Funda BİLGİLİ TETİKOĞLU, so the biggest thanks to her.

Contributions

ST performed experiments. ST and SCU wrote the manuscript. SCU designed the study.

Conflict of interest

The authors declare that there is not conflict of interest.

Compliance with Ethical Standards

Ethical approval: This article does not contain any studies with human participants or animals performed by any of the authors.

Bohnsack MT, Stuven T, Kuhn C, Cordes VC, Gorlich D (2006) A selective block of nuclear actin export stabilizes the giant nuclei of Xenopus oocytes. Nat Cell Biol 8(3):257–263. doi: 10.1038/ncb1357
Caridi CP, D'Agostino C, Ryu T, Zapotoczny G, Delabaere L, Li X, Chiolo I (2018) Nuclear F-actin and myosins drive relocalization of heterochromatic breaks. Nature 559(7712):54–. doi: 10.1038/s41586-018-0242-8
Cerutti EDL, Donnio L, Neuillet D (2019) β-Actin and Nuclear Myosin I are responsible for nucleolar reorganization during DNA Repair. Biorvix
Doery HM, Pearson JE (1964) Phospholipase B in snake venoms and bee venom. Biochem J 92(3):599–602. doi: 10.1042/bj0920599
Eden S, Rohatgi R, Podtelejnikov AV, Mann M, Kirschner MW (2002) Mechanism of regulation of WAVE1-induced actin nucleation by Rac1 and Nck. Nature 418(6899):790–793. doi: 10.1038/nature00859
Fletcher DA, Mullins RD (2010) Cell mechanics and the cytoskeleton. Nature 463(7280):485–492. doi: 10.1038/nature08908
Hamer G, Roepers-Gajadien HL, van Duyn-Goedhart A, Gademan IS, Kal HB, van Buul PP, de Rooij DG (2003) DNA double-strand breaks and gamma-H2AX signaling in the testis. Biol Reprod 68(2):628–634. doi: 10.1095/biolreprod.102.008672
Hong SJ, Rim GS, Yang HI, Yin CS, Koh HG, Jang MH, Chung JH (2005) Bee venom induces apoptosis through caspase-3 activation in synovial fibroblasts of patients with rheumatoid arthritis. Toxicon 46(1):39–45
Hu P, Wu S, Hernandez N (2004) A role for beta-actin in RNA polymerase III transcription. Genes Dev 18(24):3010–3015. doi: 10.1101/gad.1250804
Huh JE, Baek YH, Lee MH, Choi DY, Park DS, Lee JD (2010) Bee venom inhibits tumor angiogenesis and metastasis by inhibiting tyrosine phosphorylation of VEGFR-2 in LLC-tumor-bearing mice. Cancer Lett 292(1):98–110. doi: 10.1016/j.canlet.2009.11.013
Ip SW, Wei HC, Lin JP, Kuo HM, Liu KC, Hsu SC, Chung JG (2008) Bee venom induced cell cycle arrest and apoptosis in human cervical epidermoid carcinoma Ca Ski cells. Anticancer Res 28(2A):833–842
Jang MH, Shin MC, Lim S, Han SM, Park HJ, Shin I, Kim CJ (2003) Bee venom induces apoptosis and inhibits expression of cyclooxygenase-2 mRNA in human lung cancer cell line NCI-H1299. J Pharmacol Sci 91(2):95–104
Jeong YJ, Choi Y, Shin JM, Cho HJ, Kang JH, Park KK, Chang YC (2014) Melittin suppresses EGF-induced cell motility and invasion by inhibiting PI3K/Akt/mTOR signaling pathway in breast cancer cells. Food Chem Toxicol 68:218–225. doi: 10.1016/j.fct.2014.03.022
Ji J, Zhang Y, Redon CE, Reinhold WC, Chen AP, Fogli LK, Bonner WM (2017) Phosphorylated fraction of H2AX as a measurement for DNA damage in cancer cells and potential applications of a novel assay. PLoS ONE 12(2):e0171582. doi: 10.1371/journal.pone.0171582
Jo M, Park MH, Kollipara PS, An BJ, Song HS, Han SB, Hong JT (2012) Anti-cancer effect of bee venom toxin and melittin in ovarian cancer cells through induction of death receptors and inhibition of JAK2/STAT3 pathway. Toxicol Appl Pharmacol 258(1):72–81. doi: 10.1016/j.taap.2011.10.009
Jung EJ, Kim DR (2010) Control of TrkA-Induced Cell Death by JNK Activation and Differential Expression of TrkA upon DNA Damage. Mol Cells 30(2):121–125
Jung EJ, Kim DR (2011) Ectopic expression of H2AX protein promotes TrkA-induced cell death via modulation of TrkA tyrosine-490 phosphorylation and JNK activity upon DNA damage. Biochem Biophys Res Commun 404(3):841–847
Kasparek TR, Humphrey TC (2011) DNA double-strand break repair pathways, chromosomal rearrangements and cancer. Semin Cell Dev Biol 22(8):886–897. doi: 10.1016/j.semcdb.2011.10.007
Kavallaris M (2010) Microtubules and resistance to tubulin-binding agents.Nature Reviews Cancer, 10(4)
Lariviere WR, Melzack R (1996) The bee venom test: a new tonic-pain test. Pain 66(2–3):271–277. doi: 10.1016/0304-3959(96)03075-8
Leuschner C, Hansel W (2004) Membrane disrupting lytic peptides for cancer treatments. Curr Pharm Des 10(19):2299–2310. doi: 10.2174/1381612043383971
Liu X, Chen DW, Xie LP, Zhang RQ (2002) Effect of honey bee venom on proliferation of K1735M2 mouse melanoma cells in-vitro and growth of murine B16 melanomas in-vivo. J Pharm Pharmacol 54(8):1083–1089
McDonald D, Carrero G, Andrin C, de Vries G, Hendzel MJ (2006) Nucleoplasmic beta-actin exists in a dynamic equilibrium between low-mobility polymeric species and rapidly diffusing populations. J Cell Biol 172(4):541–552. doi: 10.1083/jcb.200507101
Meyer B, Voss KO, Tobias F, Jakob B, Durante M, Taucher-Scholz G (2013) Clustered DNA damage induces pan-nuclear H2AX phosphorylation mediated by ATM and DNA-PK. Nucleic Acids Res 41(12):6109–6118
Moga MA, Dimienescu OG, Arvatescu CA, Ifteni P, Ples L (2018) Anticancer Activity of Toxins from Bee and Snake Venom-An Overview on Ovarian Cancer. Molecules 23(3). doi: 10.3390/molecules23030692
Moon DO, Park SY, Heo MS, Kim KC, Park C, Ko WS, Kim GY (2006) Key regulators in bee venom-induced apoptosis are Bcl-2 and caspase-3 in human leukemic U937 cells through downregulation of ERK and Akt. Int Immunopharmacol 6(12):1796–1807
Nagelkerke A, Span PN (2016) Staining Against Phospho-H2AX (gamma-H2AX) as a Marker for DNA Damage and Genomic Instability in Cancer Tissues and Cells. Adv Exp Med Biol 899:1–10. doi: 10.1007/978-3-319-26666-4_1
Ong MS, Deng S, Halim CE, Cai W, Tan TZ, Huang RY, Yap CT (2020) Cytoskeletal Proteins in Cancer and Intracellular Stress: A Therapeutic Perspective. Cancers (Basel) 12(1). doi: 10.3390/cancers12010238
Orsolic N (2012) Bee venom in cancer therapy. Cancer Metastasis Rev 31(1–2):173–194
Orsolic N, Knezevic A, Sver L, Terzic S, Hackenberger BK, Basic I (2003) Influence of honey bee products on transplantable murine tumours. Vet Comp Oncol 1(4):216–226. doi: 10.1111/j.1476-5810.2003.00029.x
Pawlak G, Helfman DM (2001) Cytoskeletal changes in cell transformation and tumorigenesis. Curr Opin Genet Dev 11(1):41–47. doi: 10.1016/s0959-437x(00)00154-4
Putz T, Ramoner R, Gander H, Rahm A, Bartsch G, Bernardo K, Thurnher M (2007) Bee venom secretory phospholipase A2 and phosphatidylinositol-homologues cooperatively disrupt membrane integrity, abrogate signal transduction and inhibit proliferation of renal cancer cells. Cancer Immunol Immunother 56(5):627–640. doi: 10.1007/s00262-006-0220-0
Rao JY, Li N (2004) Microfilament actin remodeling as a potential target for cancer drug development. Curr Cancer Drug Targets 4(4):345–354
Redon CE, Dickey JS, Bonner WM, Sedelnikova OA (2009) gamma-H2AX as a biomarker of DNA damage induced by ionizing radiation in human peripheral blood lymphocytes and artificial skin. Adv Space Res 43(8):1171–1178. doi: 10.1016/j.asr.2008.10.011
Schmid TE, Zlobinskaya O, Multhoff G (2012) Differences in Phosphorylated Histone H2AX Foci Formation and Removal of Cells Exposed to Low and High Linear Energy Transfer Radiation. Curr Genomics 13(6):418–425. doi: 10.2174/138920212802510501
Schoenenberger CA, Buchmeier S, Boerries M, Sutterlin R, Aebi U, Jockusch BM (2005) Conformation-specific antibodies reveal distinct actin structures in the nucleus and the cytoplasm. J Struct Biol 152(3):157–168. doi: 10.1016/j.jsb.2005.09.003
Schrank BR, Aparicio T, Li YY, Chang W, Chait BT, Gundersen GG, Gautier J (2018) Nuclear ARP2/3 drives DNA break clustering for homology-directed repair. Nature 559(7712):61–. doi: 10.1038/s41586-018-0237-5
Sedelnikova OA, Pilch DR, Redon C, Bonner WM (2003) Histone H2AX in DNA damage and repair. Cancer Biol Ther 2(3):233–235. doi: 10.4161/cbt.2.3.373
Sisakht M (2017) Bee venom induces apoptosis and suppresses matrix 824 metaloprotease 2 expression in human glioblastoma cells
Sobotka AK, Franklin RM, Adkinson NF Jr, Valentine M, Baer H, Lichtenstein LM (1976) Allergy to insect stings. II. Phospholipase A: the major allergen in honeybee venom. J Allergy Clin Immunol 57(1):29–40. doi: 10.1016/0091-6749(76)90076-2
Son DJ, Lee JW, Lee YH, Song HS, Lee CK, Hong JT (2007) Therapeutic application of anti-arthritis, pain-releasing, and anti-cancer effects of bee venom and its constituent compounds. Pharmacol Ther 115(2):246–270. doi: 10.1016/j.pharmthera.2007.04.004
Sonoda E, Hochegger H, Saberi A, Taniguchi Y, Takeda S (2006) Differential usage of non-homologous end-joining and homologous recombination in double strand break repair. DNA Repair 5(9–10):1021–1029
Stucki M, Clapperton JA, Mohammad D, Yaffe MB, Smerdon SJ, Jackson SP (2005) MDC1 directly binds phosphorylated histone H2AX to regulate cellular responses to DNA double-strand breaks. Cell 123(7):1213–1226
Tojkander S, Gateva G, Lappalainen P (2012) Actin stress fibers - assembly, dynamics and biological roles. J Cell Sci 125(8):1855–1864
Tu WC, Wu CC, Hsieh HL, Chen CY, Hsu SL (2008) Honeybee venom induces calcium-dependent but caspase-independent apoptotic cell death in human melanoma A2058 cells. Toxicon 52(2):318–329
Uddin MB, Lee BH, Nikapitiya C, Kim JH, Kim TH, Lee HC, Kim CJ (2016) Inhibitory effects of bee venom and its components against viruses in vitro and in vivo. J Microbiol 54(12):853–866. doi: 10.1007/s12275-016-6376-1
Uzuner SC, Birinci E, Tetikoglu S, Birinci C, Kolayli S (2021) Distinct Epigenetic Reprogramming, Mitochondrial Patterns, Cellular Morphology and Cytotoxicity after Bee Venom Treatment. Recent Pat Anticancer Drug Discov. doi: 10.2174/1574892816666210422125058
Waterman DP, Haber JE, Smolka MB (2020) Checkpoint Responses to DNA Double-Strand Breaks. Annu Rev Biochem 89:103–133
Zheng J, Lee HL, Ham YW, Song HS, Song MJ, Hong JT (2015) Anti-cancer effect of bee venom on colon cancer cell growth by activation of death receptors and inhibition of nuclear factor kappa B. Oncotarget 6(42):44437–44451. doi: 10.18632/oncotarget.6295

Table 1. Major localisation of β-actin within the cells before and after bee venom

CELLS	Intracellular location of β-actin
CELLS	Before bee venom	After bee venom
MDA-MB-231	Cytoplasm	Nucleus (after 25µg/ml)
HePG2	Cytoplasm + nucleus	Nucleus (after 25µg/ml)
NIH3T3	Nucleus	Cytoplasm

Table 2. Major colocalisation of β-actin and γH2AX before and after bee venom in three cells

CELLS	Colocalisation
CELLS	Before bee venom	After bee venom
MDA-MB-231	N/A	Cytoplasm + nucleus
HePG2	Cytoplasm + Nucleus	Cytoplasm + nucleus
NIH3T3	Nucleus	Cytoplasm + nucleus

Download PDF

Version 1

posted

You are reading this latest preprint version

Bee venom induced the interaction of phosphorylated histone variant, γH2AX, and intracellular location of beta actin in cancer cells

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

3. Results

Discussion

Conclusion

Declarations

References

Tables

Supplementary Files

Status:

Version 1