Silica Nanoparticles Sensitize Human Multiple Myeloma Cells to Snake (Walterinnesia aegyptia) Venom-Induced Apoptosis and Growth Arrest

Background. Multiple myeloma (MM), an almost incurable disease, is the second most common blood cancer. Initial chemotherapeutic treatment could be successful; however, resistance development urges the use of higher toxic doses accompanied by hematopoietic stem cell transplantation. The establishment of more effective treatments that can overcome or circumvent chemoresistance has become a priority. We recently demonstrated that venom extracted from Walterinnesia aegyptia (WEV) either alone or in combination with silica nanoparticles (WEV+NPs) mediated the growth arrest and apoptosis of prostate cancer cells. In the present study, we evaluated the impact of WEV alone and WEV+NP on proliferation and apoptosis of MM cells. Methods. The impacts of WEV alone and WEV+NP were monitored in MM cells from 70 diagnosed patients. The influences of WEV and WEV+NP were assessed with flow cytometry analysis. Results. WEV alone and WEV+NP decreased the viability of MM cells. Using a CFSE proliferation assay, we found that WEV+NP strongly inhibited MM cell proliferation. Furthermore, analysis of the cell cycle using the propidium iodide (PI) staining method indicated that WEV+NP strongly altered the cell cycle of MM cells and enhanced the induction of apoptosis. Conclusions. Our data reveal the biological effects of WEV and WEV+NP on MM cells that enable these compounds to function as effective treatments for MM

<it>Walterinnesia aegyptia </it>venom combined with silica nanoparticles enhances the functioning of normal lymphocytes through PI3K/AKT, NFκB and ERK signaling

Abstract Background The toxicity of snake venom varies over time in some species. The venom of newborn and small juvenile snakes appears to be more potent than adults of the same species, and a bite from a snake that has not fed recently, such as one that has just emerged from hibernation, is more dangerous than one that has recently fed due to the larger volume of venom injected. Therefore, the potency of a snake's venom is typically determined using the LD50 or IC50 tests. In the present study, we evaluated the anti-tumor potential of snake venom from Walterinnesia aegyptia (WEV) on the human breast carcinoma cell line MDA-MB-231, as well as its effect on the normal mice peripheral blood mononuclear cells (PBMCs). Results This venom was used alone (WEV) or in combination with silica nanoparticles (WEV+NP). The IC50 values of WEV alone and WEV+NP in the MDA-MB-231 cells were determined to be 50 ng/ml and 20 ng/ml, respectively. Interestingly, at these concentrations, the venom did not affect the viability of normal human PBMCs. To investigate the in vivo effects of this venom further, three groups of mice were used (15 mice in each group): Group I was the control, Group II was subcutaneously injected with WEV, and Group III was injected with WEV+NP. Using flow cytometry and western blot analysis, we found that the blood lymphocytes of WEV-injected mice exhibited a significant increase in actin polymerization and cytoskeletal rearrangement in response to CXCL12 through the activation of AKT, NF-κB and ERK. These lymphocytes also showed a significant increase in their proliferative capacity in response to mitogen stimulation compared with those isolated from the control mice (P Conclusion Our data reveal the unique biological effects of WEV, and we demonstrated that its combination with nanoparticles strongly enhanced these biological effects.</p

Cellular and Molecular Mechanisms Underlie the Anti-Tumor Activities Exerted by <em>Walterinnesia aegyptia</em> Venom Combined with Silica Nanoparticles against Multiple Myeloma Cancer Cell Types

<div><p>Multiple myeloma (MM) is a clonal disease of plasma cells that remains incurable despite the advent of several novel therapeutics. In this study, we aimed to delineate the impact of snake venom extracted from <em>Walterinnesia aegyptia</em> (WEV) alone or in combination with silica nanoparticles (WEV+NP) on primary MM cells isolated from patients diagnosed with MM as well as on two MM cell lines, U266 and RPMI 8226. The IC₅₀ values of WEV and WEV+NP that significantly decreased MM cell viability without affecting the viability of normal peripheral mononuclear cells (PBMCs) were determined to be 25 ng/ml and 10 ng/ml, respectively. Although both WEV (25 ng/ml) and WEV+NP (10 ng/ml) decreased the CD54 surface expression without affecting the expression of CXCR4 (CXCL12 receptor) on MM cells, they significantly reduced the ability of CXC chemokine ligand 12 (CXCL12) to induce actin cytoskeleton rearrangement and the subsequent reduction in chemotaxis. It has been established that the binding of CXCL12 to its receptor CXCR4 activates multiple intracellular signal transduction pathways that regulate MM cell chemotaxis, adhesion, and proliferation. We found that WEV and WEV+NP clearly decreased the CXCL12/CXCR4-mediated activation of AKT, ERK, NFκB and Rho-A using western blot analysis; abrogated the CXCL12-mediated proliferation of MM cells using the CFSE assay; and induced apoptosis in MM cell as determined by PI/annexin V double staining followed by flow cytometry analysis. Monitoring the expression of B-cell CCL/Lymphoma 2 (Bcl-2) family members and their role in apoptosis induction after treatment with WEV or WEV+NP revealed that the combination of WEV with NP robustly decreased the expression of the anti-apoptotic effectors Bcl-2, Bcl_XL and Mcl-1; conversely increased the expression of the pro-apoptotic effectors Bak, Bax and Bim; and altered the mitochondrial membrane potential in MM cells. Taken together, our data reveal the biological effects of WEV and WEV+NP and the underlying mechanisms against myeloma cancer cells.</p> </div

Effect of WEV and WEV+NP on CXCL12-mediated signaling.

<p>U266 (A) and RPMI 8226 (B) cells were untreated (0) or treated with 25 ng/ml of NP, 25 ng/ml of WEV or 10 ng/ml of WEV+NP for 12 hours. Cells were then treated for 2 min with medium or 250 ng/ml CXCL12 prior to being lysed. Proteins in the cell lysates were resolved on a 7% acrylamide gel. The phosphorylation levels of AKT (p-AKT), ERK1/2 (p-ERK1/2), IκBα (p- IκBα) and PLCβ3 (p-PLCβ3) and the activation level of Rho-A (Rho-A_GTP) were corrected for the level of total β-actin on stripped blots. One representative blot for each downstream effector from 10 independent experiments is shown. Accumulated results are expressed as the mean values of normalized specific phosphorylation ± SEM from 10 separate experiments for U266 (<b>C</b>) and RPMI 8226 (<b>D</b>) cells. *P<0.05, WEV-treated vs. control; #P<0.05, WEV+NP-treated vs. control; +P<0.05, WEV+NP-treated vs. WEV-treated.</p

WEV alone or combined with NP induced apoptosis in MM cancer cells.

<p>The potential of WEV and WEV+NP to induce the apoptosis or necrosis of MM cancer cells was determined by flow cytometry based on their PI/Annexin V staining patterns. (<b>A</b>) One representative data set from 12 independent experiments is shown. (<b>B</b>) Accumulated data from 12 experiments are expressed as the mean percentage of apoptotic cells ± SEM for untreated cancer cells (0) (dotted bars), NP-treated cells (hatched bars), WEV-treated cells (closed gray bars) and WEV+NP-treated cells (closed black bars). ^*P<0.05, WEV-treated vs. NP; ^#P<0.05, WEV+NP-treated vs. NP; ⁺P<0.05, WEV+NP-treated vs. WEV-treated cells.</p

Time- and dose-dependent effect of WEV and WEV+NP treatment on cell viability.

<p>Electron microscope images of double mesoporous core-shell silica nanospheres (DMCSSs) before (<b>A</b>) and after (<b>B</b>) the venom loading. Cell viability was assessed using the MTT assay. RPMI 8226 cells (<b>C</b>), U266 cells (<b>D</b>) and normal PBMCs (<b>E</b>) were treated overnight with different concentrations (0, 1, 5, 10, 25, 50, 100 and 1000 ng/ml) of NP (open circles), WEV (gray triangles) or WEV+NP (closed black squares). RPMI 8226 cells (<b>F</b>), U266 cells (<b>G</b>) and normal PBMCs (<b>H</b>) were treated with 25 ng/ml of NP (open circles), 25 ng/ml of WEV (gray triangles) or 10 ng/ml of WEV+NP (closed black squares) for different incubation times (0, 1, 2, 6, 12, 24, 36 and 48 h). The data collected from independent experiments (n = 5) are shown, and the results are expressed as the mean percentage of viable cells ± SEM.</p

Effect of WEV and WEV+NP treatment on CXCR4 and CD54 expression in MM cells.

<p>MM cells were untreated (0) or treated with 25 ng/ml of NP, 25 ng/ml of WEV and 10 ng/ml of WEV+NP for 12 hours, and the surface expression of CXCR4 and CD54 was analyzed by flow cytometry using PE-conjugate mAbs. Histograms were gated based on viable cells, and one representative experiment is shown with the surface expression of CXCR4 (<b>A&B</b>) and CD54 (<b>C&D</b>) on U266 and RPMI 8226 cells, respectively. The results for the MFI of CXCR4 (<b>E</b>) and CD54 (<b>F</b>) expressed on primary MM cells isolated from patients with multiple myeloma that were untreated (0), or treated with NP, WEV and WEV+NP are shown (n = 12).</p

Effect of WEV and WEV+NP on the expression of Bcl-2 family members and mitochondrial membrane potential.

<p>The expression of Bcl-2 family members (anti-apoptotic proteins: Bcl-2, Bcl_XL and Mcl-1; and pro-apoptotic proteins: Bak, Bax and Bim) was monitored in the U266 and RPMI 8226 cell lines using western blot analysis. (<b>A</b>) Protein bands from one representative experiment performed using the U266 MM cell line are shown for cells that were untreated (0) or treated with NP, WEV or WEV+NP for 12 hours. (<b>B</b>) The expression of the anti-apoptotic and pro-apoptotic proteins was normalized to the total β-actin levels, and the results from 5 independent experiments performed using U266 cells are expressed as the mean ± SEM of the normalized values for the anti-apoptotic and pro-apoptotic proteins in untreated (dotted bars), NP-treated (hatched bars), WEV-treated (closed gray bars) and WEV+NP-treated (closed black bars) cells. (<b>C</b>) The expression of the anti-apoptotic and pro-apoptotic proteins was normalized to the total β-actin levels, and the results collected from 5 independent experiments performed on RPMI 8226 cells are expressed as the mean ± SEM of normalized values of the anti-apoptotic and pro-apoptotic proteins. Changes in the mitochondrial membrane potential were monitored using JC-1 and flow cytometry. (<b>D</b>) One representative experiment is shown, which discriminates between apoptotic (green dot plots) and surviving (red dot plots) U266 cells. (<b>E</b>) Accumulated data from 12 experiments are expressed as the mean percentage of apoptotic cells ± SEM for untreated cancer cells (0) (dotted bars), NP-treated cells (hatched bars), WEV-treated cells (closed gray bars) and WEV+NP-treated cells (closed black bars). ^*P<0.05, WEV-treated vs. NP; ^#P<0.05, WEV+NP-treated vs. NP; ⁺P<0.05, WEV+NP-treated vs. WEV-treated.</p