The scorpions of M. eupeus species (Fig. 1a) were nocturnally collected. Venom extraction involved anesthetizing scorpions with carbon dioxide, followed by electrical stimulation (12 V, 25 amperes) of their telsons (Fig. 1b). Collected venom was diluted in 0.85% NaCl physiological saline, lyophilized, and stored at − 20 °C for subsequent experimental procedures.

The electrophoretic profile of M. eupeus venoms was analyzed by SDS-PAGE electrophoresis [18], confirming the presence of proteins/peptides with distinct molecular weights ranging from 5 to 50 kDa in the venom composition (Fig. 1c). A specific maximum peak of the venom was observed at 280 nm with a UV–visible spectrophotometer (Fig. 1d). The quantification of total proteins in the venom was calculated as 607.5 µg/mL, referencing a BCA [17] standard curve (Fig. 1d). Subsequently, HPLC analysis was performed at 280 nm based on this observation. The HPLC analysis revealed the presence of 11 different proteins/peptides in various fractions and time points within the venom (Fig. 1e).

Colorectal cancer (CRC) stands out as a highly lethal malignancy for both genders, prompting substantial mortality annually [17, 18]. The efficacy of conventional therapeutic approaches for this cancer type remains insufficient, coupled with their frequent occurrence of adverse effects. This limitation has spurred the exploration of alternative strategies to mitigate these side effects and develop more effective treatment options [19]. Among these avenues, investigations centered around scorpion venoms, notably those rich in diverse bioactive compounds such as peptides and proteins, have garnered attention for their potential therapeutic applications [20]. The toxicity of venom can vary from region to region and even within the same species [10]. Factors such as the transport conditions of telsons used as venom sources, drying method, storage, and usage duration can also influence venom toxicity [12]. In a study by Oukkache et al. [21], characterization and lethal dose (LD50) were conducted on venoms obtained through manual and electrical stimulation methods for collecting scorpion venoms. It was found that venom extracted manually contained unwanted hemolymph-derived substances, and the venom obtained through electrical stimulation had a threefold higher LD50 value compared to manually collected venom. Characterization studies revealed that manually collected venom contained an additional 75 kDa band not present in the electrophoretic profile of electrically extracted venom. The absorption profiles at 280 nm showed that manually collected venoms had two additional absorption peaks (220–380 and 520–600 nm) not found in venoms extracted via electrical stimulation. This study demonstrated characteristic and lethal dose differences between venom extracted by manual stimulation and electrical stimulation, with the latter providing more accurate results [21]. Research is being conducted on using scorpion venom proteins/peptides in cancer treatment. Some proteins or peptides isolated from venoms specifically bind to the cancer cell membrane, affecting cancer cell migration and proliferation [22]. Maleki and colleagues conducted characterization experiments for isolating toxic peptides from the venom of Hemiscorpius lepturus, an Iranian scorpion, using gel filtration, ion exchange, and reverse-phase high-performance liquid chromatography (RP-HPLC). They identified seven bands with molecular weights between 10 and 100 kDa and a band smaller than 10 kDa from crude venom. In addition, two peaks, HL2153 and HL2155, indicative of high-purity and single-band profiles, were detected after RP-HPLC [23]. In this study, 11 different proteins/peptides from the venom of M. eupeus were analyzed using HPLC. The differences are attributed to the scorpion species and the methods used. Khoobdel et al. analyzed the proteins in M. eupeus venom using SDS-PAGE. They identified 12 bands ranging from 5 to 140 kDa using a 15% polyacrylamide gel [24]. The protein bands were detected to be between 50, 25–37, and 15–20 kDa, but no specific differences were observed below 15 kDa. These differences are due to gel percentages and the markers used.

The M. eupeus venom exhibited a dose-dependent inhibition of cell viability in hCRC cells, DLD-1 and HT-29, with calculated IC50 values of 4.32 and 7.61 µg/mL (Fig. 2a, b), respectively. However, there was no observed cytotoxic effect on the healthy human colon epithelial cells CCD18-Co (IC50 > 250 µg/mL) at these concentrations (Fig. 2a). Gerges et al. determined the IC50 value of Smp43 scorpion peptide as 4.11 µg/mL for colorectal cancer cell line (hct-116) and 62.17 µg/mL for regular colon epithelial cell line (FHC) [25]. Valizade et al. also found that M. eupeus scorpion venom had an IC50 value of 10 μg/mL for the HT-29 colon cancer cell line, while it did not affect the healthy cell line HEK-293 T [26]. Treatment with M. eupeus venom significantly reduced the colony number of both cell lines by 52.80 and 63.78%, respectively, compared to the control group (Fig. 2c, d). Besides, metastasis potency of the hCRC cells significantly (p < 0.001) following the M. eupeus venom treatment (Fig. 2e, g) by 89.24 and 62.28%, respectively (Fig. 2f, h). To clarify the death mechanism of the M. eupeus venom, the apoptotic and necrotic rates were determined using flow cytometry. After treatment with an equivalent concentration of IC50 values of M. eupeus venom, the early apoptotic rate increased by 21.7 and 3.18% in DLD-1 and HT-29 cells, respectively. The late apoptotic rate increased by 3.5 and 10.85% in DLD-1 and HT-29 cell lines, respectively. The necrotic cell rate remained unchanged in both cell lines compared to the control group (Fig. 3a, b).

Cytotoxicity, colony formation, and wound healing effects of M. eupeus venom on cell lines. a Sigmoidal graph of cell viability against log concentration of scorpion venom. b IC50 values (µg/mL) of HT-29 and DLD-1 cell lines. c Colony images of DLD-1 and HT-29 cell lines. d Graph of colony count comparing M. eupeus venom with the control group. e and g Images of in vitro wound healing experiment at 0, 24, and 48 h. f, h Graph of cell count at 0, 24, and 48 h comparing M. eupeus venom with the control group. Results are presented as the mean ± SDV of three independent experiments. *p < 0.01, **p < 0.001, and ***p < 0.0001

Effects of M. eupeus venom on apoptosis in DLD-1 and HT-29 cell lines. a-1 and b-1 Flow cytometry images of cells in the control group (Non-treated). a-2 and b-2 Flow cytometry images of cells treated with M. eupeus venom (Q1: necrotic cells; Q2: late apoptotic cells; Q3: live cells; Q4: early apoptotic cells). a-3 and b-3 Ratio of necrosis, early apoptosis, and late apoptosis compared to the control group for M. eupeus venom. Results are presented as the mean ± SDV of three independent experiments. **p < 0.001, and ***p < 0.0001

Valizade et al. used MTT assays to evaluate the impact of M. eupeus venom on the HT-29 colon cancer cell line [26]. Flow cytometry was used for apoptotic and colony formation analyses. Results showed dose-dependent cytotoxicity with a significant reduction in cell viability at doses above three µg/mL. In addition, flow cytometry analysis indicated that the venom-induced late apoptosis in HT-29 cells compared to early apoptosis [26]. In this study, cytotoxicity studies were performed using the Alamar Blue method, which is more specific and sensitive than MTT. The scorpion venom showed significant cytotoxicity against HT-29 and DLD-1 colon cancer cell lines, while no cytotoxic effect was observed on the colon epithelial cell line CCD18-Co. Apoptosis analysis revealed lower early apoptosis rates in HT-29 and early apoptotic induction in DLD-1 cells. Colony formation experiments showed decreased colony numbers and sizes in HT-29 and DLD-1 cell lines. In silico analyses identified 10 peptides/proteins in M. eupeus scorpion venom (Table 1). These peptides were potassium channel inhibitors, suggesting that M. eupeus scorpions exert cytotoxic effects by inhibiting K + channels in cells. Furthermore, Gandomkari et al. demonstrated the cytotoxic effect of the recombinant MeICT (rMeICT) peptide isolated from M. eupeus venom on glioma cells, with IC50 values of 3 and 5 µM, and in vitro wound healing assays showed wound closure percentages of 58 and 22%, respectively [27]. These findings highlight the potential of rMeICT peptide as an agent targeting gliomas [26]. In this study, in vitro metastasis experiments showed that M. eupeus venom inhibited metastasis in both cell lines. However, the HT-29 cell line, with higher adhesive properties, exhibited relatively lower metastasis rates than DLD-1. The different molecular mechanisms and increased protein expressions in the metastatic DLD-1 cells, inhibited by scorpion venom, explain the migration rates between the cell lines.

Treatment with M. eupeus venom altered the mRNA expressions of genes involved in apoptotic pathways (Figure S1). The venom treatment increased mRNA expression of 19 genes (ATM, BAG3, BCL2A1, BCL2L11, BIRC5, BNIP3, CASP1, CASP12, CASP3, CIDEB, DAPK2, DFFB, TNF, TNFRSF10A, TNFRSF10B, TNFRSF10C, TNFRSF13, TNFRSF9) and decreased mRNA expression of 22 genes (BCL2, BCL2L2, BIRC1, BIRC2, BIRC6, BOK, BRCC45, CARD4, CASP8, CHEK2, FAS, GADD45A, MCL1, MYD88, RIPK2, RPA3, TNFRSF13B, TNFRSF15, TRAF2, TRAF3, TRIP) in apoptotic pathway in DLD-1 cells (Fig. 4a; Table S1). On the other hand, mRNA expression of 12 genes (APAF1, ATM, BAG3, BAG4, BAK1, CASP12, CASP3, CASP5, CASP7, TRAF3, TRAF4, TRIP) was elevated, while 44 gene’s mRNA expression (BCL2A1, BCLX, BFAR, BIK, BIRC1, CARD4, CASP2, CASP6, CASP8, CASP9, CD40L, CHEK1, CIDEA, CIDEB, CRADD, DAPK1, DAPK2, DFFA, DFFB, FAS, FASLG, GADD45A, HRK, LTBR, MCL1, MYD88, RIPK2, RPA3, TANK, TNFRSF10, TNFRSF10A, TNFRSF10B, TNFRSF10C, TNFRSF11, TNFRSF11B, TNFRSF13, TNFRSF13B, TNFRSF15, TNFRSF4, TNFRSF8, TNFRSF9, TP53, TRAF1, TRAF5) was decreased following venom treatment in HT-29 cells (Fig. 4b; Table S1). mRNA studies have shown that M. eupeus venom treatment significantly affects the expression of genes in the apoptotic pathway (Fig. 4c, d). In DLD-1 cells, the mRNA expressions of Bax, Caspase-3, and Caspase-12 significantly increased by 3.7-fold, 12.5-fold, and 1.8-fold, respectively (p < 0.0001). In contrast, Bcl-2 mRNA expression decreased by 68.75%. In HT-29 cells, the mRNA expressions of Bax, Caspase-3, Caspase-9, and Caspase-12 significantly increased by 3.01-fold, 2.94-fold, 1.78-fold, and 3.8-fold, respectively (p < 0.0001). Bcl-2 mRNA expression was decreased by 77% compared to non-treated cells.

Effects of M. eupeus venom on genes and proteins involved in apoptosis. a, b Alteration in expression levels of 96 genes involved in the apoptosis panel following the treatment with M. eupeus scorpion venom. c, d Alteration in expression levels of genes BcL-2, Bax, caspase-3, caspase-9, and caspase-12, which play an important role in apoptosis, in DLD-1 and HT-29 cell lines compared to the control group. e, f Representative Immunoblot of proteins Bax, BcL-2, and GAPDH. g, h Analysis of band density of protein expression of Bax and BcL-2 compared to the control group relative to the reference protein (GAPDH). Results are presented as the mean ± SDV of three independent experiments. *p < 0.01, **p < 0.001, and ***p < 0.0001

Correlated with mRNA expressions, the protein expressions were also altered following the M. eupeus venom (Fig. 4e, f). The protein expressions of key proteins in the apoptotic pathway, Bax, Bcl-2, and caspase-3, were analyzed (Fig. 4g, 4h). In DLD-1 cells, venom treatment significantly (p < 0.0001) increased Bax and caspase-3 protein expressions by 1.7-fold and 1.5-fold, respectively. On the other hand, venom treatment decreased Bcl-2 protein expression by 32% (p < 0.001) compared with the control group. In HT-29 cells, no alteration was detected in Bax protein expression following venom treatment. However, caspase-3 protein expression was significantly (p < 0.05) increased by 1.56-fold, and Bcl-2 protein expression was decreased by 50% (p < 0.0001).

Treatment with M. eupeus venom changed the mRNA expressions of genes involved in human colorectal cancer (hCRC) (Fig. S2). The venom treatment increased mRNA expression of 16 genes (BMP4, CEACAM1, HHAT, INHBA, ITGA4, PIK3CA, PRKCA, PRKCB, SMAD4, SOX9, STAT3, TYMS, VEGFA, VIM, WIF1, WNT5) and decreased mRNA expression of 23 genes (ALX4, APC, AXIN1, AXIN2, BGN, CDKN2A, CEACAM5, DLL4, DVL2, EGFR, FLT1, FZD1, FZD3, GADD45B, GLI1, IGFBP7, MAPK3, MAPK8, MGMT, MYC, NOTCH2, SFRP2, TGFB2) in hCRC progression pathway in DLD-1 cells (Fig. 5a; Table S2). On the other hand, mRNA expression of 57 genes (BMP4, ADAM17, AKT1, ALX4, APC, AXIN1, AXIN2, BGN, BMP1, BMP2, BMP4, BMPR1, BRAF, CDKN2A, CDX2, CEACAM1, CEACAM5, CHRD, DHH, DLL1, DLL4, DVL1, DVL2, EGFR, FAP, FLT1, FZD1, GADD45B, GLI1, HHAT, IGFBP7, ITGA4, JAG1, JAG2, MAPK1, MAPK8, MYBL2, NEUROG1, NGFR, NOTCH1, NOTCH2, PRKCA, PTCH1, PTEN, RASSF2, SLC5A8, SMAD4, SMAD3, SMAD4, SMO, SOX9, TP53, VEGFA, VIM, WIF1, WNT1) was elevated, while one gene’s mRNA expression (GLI-2) was decreased following venom treatment in HT-29 cells (Fig. 5b; Table S2). mRNA studies have shown that M. eupeus venom treatment significantly affects the mRNA expressions of key genes in the hCRC pathway, including APC, KRAS, TP53, PIK3CA, SMAD4, BRAF, PTEN, and CTNNB1 (Fig. 5c; Fig. 5d). In DLD-1 cells, the mRNA expressions of TP53, PIK3CA, PTEN, and SMAD4 were significantly increased by 6.5-fold, 1.37-fold, 1.7-fold, and 4.5-fold, respectively (p < 0.0001), while KRAS mRNA expression decreased by 35.29%. In HT-29 cells, the mRNA expressions of APC, TP53, SMAD4, and PTEN significantly increased by 1.86-fold, 2.92-fold, 3.14-fold, and 2.2-fold, respectively (p < 0.0001). Bcl-2 mRNA expression was decreased by 77% compared to non-treated cells. Correlated with the mRNA studies, protein expression studies have also elevated in hCRC (Fig. 5e, f). In DLD-1 cells, venom treatment significantly (p < 0.0001) increased p53 protein, SMAD-4, NF-κB, and PTEN protein expressions by 2.6-fold, 3.1-fold, 3.0-fold and 1.8-fold, respectively. Besides, these protein expressions in HT-29 cells were also significantly (p < 0.0001) elevated as 3.4-fold, 2.8-fold, 1.2-fold, and 2.6-fold, respectively.

Effects of M. eupeus venom on genes and proteins involved in human colorectal carcinoma progression. a, b Alteration in mRNA expression levels of 96 genes involved in the development of colon cancer following the treatment of M. eupeus venom. c, d Alteration in expression levels of genes NF-κB, TP53, SMAD4, APC, BRAF, KRAS, and MLH-1, which play a role in the human colorectal carcinoma progression, in DLD-1 and HT-29 cell lines compared to the control group. e, g Representative Immunoblot of NF-κB, P53, and GAPDH proteins. f, h Analysis of band density of protein expression of NF-κB and P53 compared to the control group relative to the reference protein (GAPDH). Results are presented as the mean ± SDV of three independent experiments. *p < 0.01, and ***p < 0.0001

Apoptosis, the process of programmed cell death, plays a crucial role in normal development, tissue maintenance, and the elimination of damaged cells. The apoptosis pathway can be divided into extrinsic and intrinsic pathways. The extrinsic pathway, mediated by death receptors such as Fas, tumor necrosis factor (TNF) receptors, and TNF-related apoptosis-inducing ligand (TRAIL) receptors, is triggered by external or internal stimuli [28]. The binding of ligands to these death receptors activates caspase-8, activating executioner caspases-3/6/7, promoting apoptosis. The decrease in Fas and caspase-8 observed in both cell lines indicates the venom's effect on the apoptosis mechanism [29]. It was demonstrated an increase in caspase-3 and a decrease in Fas and caspase-8 in both DLD-1 and HT-29 cell lines, indicating that the venom induces cell death via the extrinsic apoptosis pathway. In addition, western blot analysis confirmed the increase in caspase-3, further proving the venom's impact on apoptosis.

RIP (Receptor Interacting Protein) and NF-κB are crucial in cell signaling and immune response. RIP is part of a protein complex involved in cell death signaling, and its activation can lead to apoptosis or an inflammatory response during cell stress or damage. NF-κB is a transcription factor that regulates the expression of specific genes. RIP affects the NF-κB signaling pathway, regulating cellular responses such as cell death [30]. Decreased RIP gene expression led to an increase in NF-κB gene expression in both DLD-1 and HT-29 cell lines. Western blot analysis also showed increased NF-κB protein expression in both cell lines, indicating the venom's effect on these proteins. The intrinsic apoptosis pathway is triggered by chemotherapy, radiotherapy, and stress. The functional outcome of pro-apoptotic signaling in the inherent pathway involves mitochondrial membrane disruption and the release of cytochrome c into the cytoplasm, forming the apoptosome with apoptotic protease activating factor 1 (APAF1) and pro-caspase-9. This complex activates initiator caspase-9, which then cleaves and activates caspases-3/6/7, resulting in apoptosis. The apoptosis panel results showed that scorpion venom did not affect intrinsic apoptosis in the DLD-1 cell line. However, increased APAF1 gene expression and decreased caspase-9 gene expression in the HT-29 cell line suggested that the venom induces cell death via intrinsic apoptosis through caspase-3.

The development of colorectal cancer involves chromosomal abnormalities, gene mutations, and epigenetic changes in genes that regulate proliferation, differentiation, apoptosis, and angiogenesis. The Wnt signaling pathway, involving APC and Axin1/2, plays a role in cell proliferation, differentiation, and apoptosis. Overactivation of the Wnt pathway can lead to uncontrolled cancer cell growth and tumor formation [31]. Normally, the Wnt/β-catenin signaling pathway involves APC forming a complex with Axin to degrade the oncogene β-catenin, preventing it from binding to transcription factors in the nucleus and suppressing the expression of genes like c-myc or cyclin D1. In colon cancer, mutated APC cannot form a complex with Axin, allowing β-catenin to activate c-myc or cyclin D1, leading to uncontrolled cell growth. This study showed that APC decreased in the DLD-1 cell line, indicating no effect from the venom, while increases in APC and β-catenin in the HT-29 cell line indicated the venom's impact on the WNT signaling pathway. While this study provides correlative gene expression data suggesting the potential involvement of these pathways, we acknowledge the need for direct experimental validation. The Wnt/β-catenin pathway has been well-documented in colorectal cancer and is known to play a critical role in tumor initiation, progression, and metastasis. Previous studies have shown that aberrant activation of the Wnt/β-catenin pathway can drive cell proliferation, inhibit differentiation, and promote cancer cell survival [32, 33]. The gene expression data of current study suggests that the upregulation of key components of this pathway are consistent with these findings and indicate that this pathway may contribute to the observed effects of M. eupeus venom. Similarly, NF-κB signaling is a central regulator of inflammation, immune responses, apoptosis, and cancer progression. Dysregulation of NF-κB has been linked to colorectal cancer pathogenesis, chemoresistance, and metastasis [34]. In particular, studies have shown that the activation of NF-κB can promote tumor growth and resistance to chemotherapy in colorectal cancer [35]. This study’s gene expression analysis aligns with these observations, suggesting the potential involvement of NF-κB signaling in the cytotoxic effects induced by the venom. TP53 is a tumor suppressor gene that increases in response to DNA damage, halting cell division, inducing apoptosis and senescence, and facilitating DNA repair by binding to the promoter regions of genes involved in these processes [36]. The increase in TP53 gene expression was further confirmed that the venom induces cell death via extrinsic apoptosis in DLD-1 and intrinsic and extrinsic apoptosis in HT-29 cell lines. In the TGFβ signaling pathway, Smad4 is crucial in promoting metastasis and proliferation in colon cancer cells [37]. Tumor suppressor Smad proteins are activated upon binding to TGFβ receptors and translocate to the nucleus to regulate the expression of specific genes, thereby inhibiting metastasis and uncontrolled cell proliferation [38]. It has been demonstrated increased gene expression of Smad4 in both DLD-1 and HT-29 cell lines. This indicates that M. eupeus scorpion venom inhibits cell metastasis and growth. KRAS is a gene and protein complex commonly known as an oncogene called Kirsten Rat Sarcoma Virus Proto-Oncogene. The KRAS gene stimulates cellular growth and division by binding to receptors on the cell surface. When mutated, the KRAS gene can lead to uncontrolled cellular growth and division, contributing to the development of cancerous tumors [39]. It has observed a decreased KRAS gene expression in DLD-1 and HT-29 cell lines. This suggests that M. eupeus scorpion venom inhibits cell growth. The BRAF gene is a proto-oncogene involved in the MAPK (Mitogen-Activated Protein Kinase) signaling pathway, which regulates intracellular signal transduction. This pathway is crucial for cellular growth, proliferation, differentiation, and survival. Mutations in the BRAF gene can lead to the overactivation of the MAPK signaling pathway, resulting in uncontrolled cell growth and division [40]. M. eupeus scorpion venom did not significantly affect the BRAF gene. PIK3CA, a tumor suppressor gene, encodes the catalytic alpha subunit of phosphatidylinositol 3-kinase (PI3K). This gene is a key component of the PI3K/AKT/mTOR signaling pathway, which regulates cell growth, proliferation, differentiation, survival, and metabolism. Overactivation or dysregulation of this pathway plays a role in cancer development [41]. M. eupeus scorpion venom increased the expression level of this gene in the DLD-1 cell line but did not affect the HT-29 cell line. PTEN is a tumor suppressor gene and protein complex involved in cell growth, proliferation, survival, and metabolism. The PTEN gene controls cell growth by regulating intracellular signal transduction. Mutations in the PTEN gene or the functional loss of the PTEN protein can lead to uncontrolled cell growth and division, contributing to the development of cancerous tumors [42]. Our studies have found that M. eupeus scorpion venom activates this gene in DLD-1 and HT-29 cell lines, preventing cancer formation. CTNNB1 is associated with the beta-catenin protein, which regulates cell adhesion and communication. Mutations in the CTNNB1 gene or abnormal activation of the beta-catenin protein can lead to the overactivation of several signaling pathways that regulate cell growth and division, contributing to cancer development and progression [43]. This study observed that M. eupeus scorpion venom activated this tumor suppressor gene in the DLD-1 cell line, while no change was observed in the HT-29 cell line. The SOX9 gene, a transcription factor, can act as either a proto-oncogene or a tumor suppressor gene, depending on the type of cancer. SOX9 regulates the tumor microenvironment, maintains epithelial barrier integrity, and preserves undifferentiated stem cells for tissue renewal in the intestinal epithelium. Studies have shown that the overexpression of SOX9 in colon cancer cells inhibits cell proliferation, while a decrease in SOX9 expression increases the proliferation of human HT-29 colon cancer cells [44]. The studies have found that SOX9 acts as a tumor suppressor gene, with increased expression levels in both DLD-1 and HT-29 cell lines. Additionally, SOX9 influences cell migration and invasion through the Wnt/β-catenin signaling pathway [45]. GLI-2, a prognostic marker in colon cancer, showed decreased expression in both cell lines [46]. Similarly, VIM, a gene expressed at high levels in metastatic tumors, showed increased expression in DLD-1 and HT-29 cell lines [47].

The conformation of a chemical agent to a drug candidate takes an extended period due to the toxic nature of the compound [48]. Captopril, ziconotide, atracurium, and eptifibatide are FDA-proven drugs formulated from venom toxins [49]. Vejovine is also an antimicrobial peptide purified from the venom of the scorpion V. mexicanus smithi. This peptide also showed hemolytic activity against human erythrocytes with 100 mM HC50 [50]. The molecular studies proved that M. eupeus venom has the potential to deal with human colorectal carcinoma. Hence, the active compounds in the venom may be further investigated for their potential against hCRC.