Neosetophomone B induces apoptosis in multiple myeloma cells via targeting of AKT/SKP2 signaling pathway

Abstract Multiple myeloma (MM) is a hematologic malignancy associated with malignant plasma cell proliferation in the bone marrow. Despite the available treatments, drug resistance and adverse side effects pose significant challenges, underscoring the need for alternative therapeutic strategies. Natural products, like the fungal metabolite neosetophomone B (NSP‐B), have emerged as potential therapeutic agents due to their bioactive properties. Our study investigated NSP‐B's antitumor effects on MM cell lines (U266 and RPMI8226) and the involved molecular mechanisms. NSP‐B demonstrated significant growth inhibition and apoptotic induction, triggered by reduced AKT activation and downregulation of the inhibitors of apoptotic proteins and S‐phase kinase protein. This was accompanied by an upregulation of p21Kip1 and p27Cip1 and an elevated Bax/BCL2 ratio, culminating in caspase‐dependent apoptosis. Interestingly, NSP‐B also enhanced the cytotoxicity of bortezomib (BTZ), an existing MM treatment. Overall, our findings demonstrated that NSP‐B induces caspase‐dependent apoptosis, increases cell damage, and suppresses MM cell proliferation while improving the cytotoxic impact of BTZ. These findings suggest that NSP‐B can be used alone or in combination with other medicines to treat MM, highlighting its importance as a promising phytoconstituent in cancer therapy.


| INTRODUCTION
Multiple myeloma (MM) is a rare cancer of the blood and bone marrow that triggers various symptoms, such as anemia, hypercalcemia, bone destruction, abnormal bleeding, and renal failure (Firth, 2019).While therapy has improved MM patients' life quality and expectancy, drugresistant clones often render most patients incurable, leading to decreased survival rates (Thorsteinsdottir et al., 2018;Yang & Lin, 2015).
Thus, there is a pressing need for new treatments to combat this devastating disease (Li et al., 2021;Liu et al., 2021;Yang & Lin, 2015).
Natural compounds have played a significant role in developing anticancer drugs, with examples such as actinomycin D, etoposide, docetaxel, mitomycin C, bleomycin, paclitaxel, and vincristine (Kinghorn et al., 2016(Kinghorn et al., , 2009).Fungi's secondary metabolites have gained much attention as a potential source of new cytotoxic scaffolds, with meroterpenoids being structurally diverse and isolated from various sources such as fungi, marine organisms, animals, and plants (Kinghorn et al., 2016;Zhao et al., 2021).Neosetophomone B (NSP-B), a meroterpenoid fungal secondary metabolite isolated from an unidentified Neosetophoma sp.(strain MSX50044), is cytotoxic in solid cancer cell lines at micromolar doses (El-Elimat et al., 2019).However, the mechanism underlying NSP-B-mediated cytotoxicity remains unclear.The phosphatidylinositol 3-kinase/AKT (PI3K/AKT) signaling pathway and its downstream effectors play a critical role in oncogenesis and are frequently activated in many cancers (Cantley, 2002;Geyer et al., 2018).AKT activation stimulates antiapoptotic signaling by activating NF-kappa B, Bad, GSK3, forkhead box 1, and inhibitors of apoptotic proteins (IAPs; Cardone et al., 1998;Cross et al., 1995;Datta et al., 1997;Uddin et al., 2008).S-phase kinase-associated protein 2 (SKP2) is a component of the SCF (SKP1-cullin-F-box) E3 ubiquitin ligase complex, which targets proteins for degradation.The role of SKP2 in cellular processes like cell cycle progression, apoptosis, cellular proliferation, and cellular differentiation makes it a significant player in tumorigenesis and cancer progression (Uddin et al., 2016).Numerous studies have shown that SKP2 is overexpressed in many human cancers, including prostate, breast, lung, colorectal, and gastric cancers, as well as lymphomas, melanomas, and sarcomas.Overexpression of SKP2 is associated with poor prognosis in many of these cancers (Gstaiger et al., 2001;Radke et al., 2005).SKP2 plays a critical role in the regulation of the G1/S phase transition of the cell cycle by promoting the degradation of cell cycle inhibitors such as p27Kip1 and p21Cip1 (Tsvetkov et al., 1999).Thus, aberrant expression of SKP2 can lead to uncontrolled cell proliferation, a key characteristic of cancer.SKP2 can degrade the tumor suppressor p53 and other proteins involved in the inhibition of cell growth (Wei et al., 2004).Inhibiting SKP2 function could stabilize these tumor suppressor proteins and inhibit cancer cell growth.SKP2 can influence the epithelial-mesenchymal transition, a process through which cancer cells acquire invasive and metastatic properties.Inhibition of SKP2 could potentially suppress these processes (Lin et al., 2010).SKP2 has been implicated in resistance to chemotherapy, and targeting SKP2 could potentially restore the sensitivity of cancer cells to these treatments (Chan et al., 2010).The AKT/SKP2 signaling pathway is known to play a crucial role in cell survival, proliferation, and apoptosis (Kulinski et al., 2018).Overactivation of this pathway is seen in several cancer types, including MM, and it promotes disease progression and resistance to treatment (Li et al., 2006).
AKT activation leads to phosphorylation and stabilization of SKP2, which in turn promotes cell cycle progression and survival (Chan et al., 2012).
Given the evidence suggesting an aberrant AKT/SKP2 pathway in MM and the antiproliferative effect of NSP-B, it is plausible to hypothesize that NSP-B may exert its therapeutic effect via the AKT/SKP2 pathway.This could involve NSP-B-induced downregulation of AKT phosphorylation and SKP2 expression, which may inhibit cell proliferation and induce apoptosis in MM cells.
In this study, we aimed to investigate NSP-B's potential to inhibit tumor growth in MM cell lines.Our results demonstrate that NSP-B treatment of MM cells decreased cell viability by inducing apoptosis.
Furthermore, NSP-B suppressed the AKT/SKP2 signaling axis and its downstream substrate molecules, including IAPs.Interestingly, NSP-B enhanced the anticancer effects of bortezomib (BTZ) on MM cells.

| Isolation of the fungal compound NSP-B
NSP-B was isolated from Neosetophoma sp.(strain MSX50044).Solidphase cultures of Neosetophoma sp. were grown on rice and extracted using CHCl 3 −MeOH (1:1).The crude extracts were fractionated using normal-phase flash chromatography and purified using preparative highperformance liquid chromatography.High-resolution electrospray ionisation mass spectroscopy and nuclear magnetic resonance were used to determine the structure of NSP-B.X-ray crystallography diffraction was also used to corroborate the structure.The purity of NSP-B was determined to be greater than 97% using ultra-performance liquid chromatography chromatograms (El-Elimat et al., 2019).

| Measurement of apoptosis
NSP-B-induced apoptosis was confirmed using the Annexin V/propidium iodide (PI) dual staining assay.Briefly, 2 × 10 6 cells were treated with NSP-B for 48 h before being harvested, rinsed in ice-cold PBS, and stained with Annexin V-FITC and PI in 1X binding buffer for 20 min.Using flow cytometry and the BD LSR Fortessa analyzer (BD Biosciences), the cells were categorized as live (Annexin FITC negative, PI negative), early apoptotic (Annexin FITC positive, PI negative), late apoptotic (Annexin FITC positive, PI positive), or necrotic (Annexin FITC negative and PI positive).The apoptosis percentage was computed by combining the early and late apoptotic cell percentages (Prabhu et al., 2021).

| Cell cycle analysis
U266 and RPMI8226 cells were treated with NSP-B for 48 h.The cells were then exposed for 30 min at 37°C to Hoechst 33342 fluorescent stain in a complete medium.Flow cytometry analyzed the cell cycle distribution using the BD LSR Fortessa analyzer (BD Biosciences), as previously mentioned (Al-Tamimi et al., 2022).

| Immunoblotting technique
U266 and RPMI8226 cells were treated with NSP-B, BTZ, and z-VAD-fmk for 48 h as previously described.Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate proteins and transferred to a polyvinylidene difluoride (PVDF) membrane (Biorad).Cell lysates were prepared, and the protein quantification was performed using the ND-1000 spectrophotometer (Nanodrop Technologies, Thermoscientific).A total of 50-100 µg of protein were loaded and separated in SDS-PAGE and transferred into a PVDF membrane (Biorad).Several antibodies were utilized for immunoblotting, and a ChemiDoc system (Bio-Rad) was used to generate and visualize the blots) (Akhtar et al., 2022).

| Flow cytometric analysis of active caspase 3 and cleaved PARP
After 48 h of treatment with NSP-B at varying lower doses (0.1, 0.5, 1, 2.5, and 5 µM), cells were fixed and permeabilized using the BD Cytofix/Cytoperm plus fixation and permeabilization solution kit.Anti-active Caspase-3-BV605 and PARP Cleaved Form-AF700 antibodies were used to stain 0.3 × 10 6 cells in HBS Hank's balanced salt solution (HBSS) for 30 min.The cells were resuspended in the same solution after being rinsed once more with HBS.Flow cytometry using a BD LSR Fortessa analyzer was used to determine the percentage of cells with activated caspase-3 and cleaved PARP (Iskandarani et al., 2016).
2.10 | Quantitation of DNA double-strand breaks MM cells were treated with NSP-B with lower doses for 48 h.
After incubation, the cells were fixed and permeabilized using a BD Cytofix/Cytoperm plus fixation and permeabilization solution kit, as per the manufacturer's instructions.H2AX (pS139)-Alexa Fluor 647 antibody was used to stain 0.3 × 10 6 cells in HBSS for 30 min at room temperature.Using a BD LSRFortessa analyzer, DNA damage was measured by flow cytometry and quantified (Iskandarani et al., 2016).

| Measurement of mitochondrial membrane potential (MMP)
For 48 h, U266 and RPMI8226 cells were treated with NSP-B at various doses.The membrane-permanent JC-1 was used to stain the cells, and flow cytometry on a BD LSR Fortessa analyzer (BD Biosciences) was used to measure the potential of the mitochondrial membrane (Prabhu et al., 2017).

| Statistical analysis
One-way analysis of variance and Tukey's multiple comparison test were used to compare between the groups.The data were Denisitometric analysis was carried out for all the western blots using ImageJ software.

| NSP-B reduces the viability and induces apoptosis in MM cells
We investigated the potential of NSP-B to inhibit the viability of U266 and RPMI8226 MM cells.The cells were treated with various concentrations of NSP-B (0.5, 1, 2.5, 5, 10, 20, and 40 µM) for 48 h, and cell viability was measured using the CCK-8 assay.We observed a dose-dependent reduction in cell growth in both cell lines, with most concentrations resulting in statistically significant growth suppression (Figure 1a).To determine if this reduction in cell viability was due to cell death, live/dead assays were performed.The proportion of dead cells increased in a dose-dependent manner following NSP-B treatment (Figures 1b and S1b).We also evaluated whether NSP-B induced apoptosis in MM cells by treating U266 and RPMI8226 cells with experimental doses of NSP-B for 48 h, and then staining with Annexin V.
Significant apoptosis was observed at doses of 1 μM and higher (Figure 1c).The combined apoptosis percentages for U266 cell line are 36.63%at 1 μM, 37.85% at 5 μM, and 43.62% at 10 μM, and for RPMI8226 cell line are 70.4% at 1 μM, 72.7% at 5 μM, and 75.1% at 10 μM.Similarly, significant apoptosis was observed at doses 0. cycle distribution, and we found that the percentage of cells in the SubG0/G1 phase was significantly higher after 48 h of NSP-B treatment compared to controls.The SubG0 fractions for the U266 cell line are 1.72% for untreated cells, 29.6% at 1 μM, 29.3% at 5 μM and 32.5% at 10 Μm of NSP-B, and for RPMI8226 cell line are 7.10% for untreated cells, 25.9% at 1 μM, 26.1% at 5 μM, and 27.0% at 10 Μm of NSP-B (Figure 1d).Moreover, NSP-B treatment led to a dose-dependent upregulation of p-H2AX expression, a hallmark of double-stranded breaks (Figure 1e), which was further confirmed in western blot analysis and flow cytometry in lower doses (Figures S2a-c and S5c,d).

| NSP-B activates intrinsic and extrinsic apoptotic pathways in MM cells
In this study, we investigated the effects of NSP-B on caspase-8 signaling and mitochondrial integrity in MM cells (Kuttikrishnan et al., 2019).Our results indicate that NSP-B likely activates the extrinsic apoptotic signaling pathway and triggers Bid truncation via caspase-8 activation, leading to increased apoptosis in U266 and RPMI8226 cells (Figure 2a).
We also found that NSP-B treatment upregulated Bax expression and downregulated Bcl2 expression in these cells, which may contribute to the proapoptotic effects of NSP-B (Figure 2a).Furthermore, we observed a decrease in MMP in NSP-B-treated cells, which may suggest that NSP-B modulates mitochondrial integrity (Figure 2b).Additionally, NSP-B treatment led to the release of cytochrome c from the mitochondria, which triggered the activation of caspase-9, -3, and PARP in a dosedependent manner in experimental doses (Figure 2c  the cyclin-dependent kinases CDK-6 and -4, was observed to decrease dose-dependent (Figure 4).

| Cotreatment with NSP-B and BTZ enhances cytotoxicity in MM cells
We proceeded to identify the subtoxic doses of NSP-B and BTZ for their anticancer efficacy on myeloma cells.BTZ, a Food and Drug Administration (FDA)-approved proteasome inhibitor, is commonly used to treat refractory MM (Iskandarani et al., 2016).However, its effectiveness is reduced by developing resistance and relapse (Murray et al., 2014;Zaal et al., 2017).We treated U266 and RPMI8226 cells with varying doses of NSP-B and BTZ to determine their cytotoxic effects.Interestingly, subtoxic doses of NSP-B (0.5 μM) and BTZ (10 nM for U266 and 2.5 nM for RPMI8226) significantly decreased cell viability in both cell lines after 48 h, as determined by a live and dead assay kit (Figures 5a and S5a).
Furthermore, the combination treatment activated caspase-3 and PARP, resulting in apoptosis, as evidenced by Western blot analysis results (Figure 5b).

| DISCUSSION
Over the years, natural compounds have emerged as a promising approach for treating various malignancies, including cancer.These bioactive compounds act by targeting different signaling molecules and pathways, and growing evidence shows they can boost chemotherapy's effectiveness (Boulos et al., 2019;Mondal et al., 2019;Yuan et al., 2017).
Our study found that NSP-B induces apoptosis, also known as programmed cell death, in leukemic cells by triggering mechanisms such as the extrinsic receptor and intrinsic mitochondrial pathways (Elmore, 2007;Vasilikos et al., 2017).By increasing the Bax/Bcl2 ratio, NSP-B triggers the release of cytochrome c into the cytoplasm, which produces an apoptosome complex that activates procaspase 9 and provides the PARP cleavage signal via activated caspase-3 (Duriez & Shah, 1997).NSP-B dose-dependently cleaved caspase-9, caspase-3, and PARP, initiating the intrinsic caspase-mediated apoptosis pathway, and induced the DNA damage marker H2AX, leading to DNA degradation and apoptosis (Oben et al., 2017).
Overexpression of SKP2 and high levels of AKT activity have been observed in numerous cancers (Kuttikrishnan, Prabhu, et al., 2022).
Treatment with NSP-B leads to the dephosphorylation of AKT and downregulation of SKP2, increasing the expression of p27, p21, and ubiquitin in MM cells.These findings suggest that NSP-B's anticancer effects on MM cells are due to its selective inhibition of the AKT/ SKP2 signaling pathway.Inhibition of AKT activity using AKT siRNA leads to decreased SKP2 expression, caspase activity, and ultimately cell death.Cyclins and CDKs play a crucial role in regulating cell cycle progression.Vermeulen et al. (2003).In this study, we investigated the effect of NSP-B on the expression of cyclins and CDKs, which are commonly overexpressed in various cancers (Shapiro, 2006).Our results showed that treatment with NSP-B led to a dose-dependent decrease in CDK-6 and -4 expression and cyclins B1 and D1 in U266 and RPMI8226 cells.In addressing drug resistance in cancer treatment, emerging research posits a potential solution in repurposing FDA-approved chemotherapeutic agents for use against various malignancies when amalgamated with other drugs or inhibitors (Chakravarty et al., 2022;Goel et al., 2021;Ramisetty et al., 2023;Scuoppo et al., 2019).Collectively, these studies illuminate the

2. 5 |
Live/dead assay U266 and RPMI8226 cells were treated with escalating doses of NSP-B, and then incubated for 48 h.The live and dead stain was prepared by adding 5 µL of calcein AM (Component A) and 20 µL of ethidium homodimer-1 (Component B) to 10 mL of phosphate buffer saline (PBS).The cells were stained with this solution for 15-30 min at room temperature in the dark.The EVOS FLoid Cell Imaging statistically analyzed using GraphPad Prism software (version 5.0 for Windows; GraphPad Software Inc., http://www.graphpad.com).The data are presented as a mean standard deviation.Values of *p ≤ .05,**p ≤ .01,and ***p ≤ .001are statistically significant.
5 μM and higher (Figures S1a,b and S5a,b).Flow cytometry was used to analyze cell F I G U R E 1 Effect of NSP-B on cell viability of MM cells.(a) NSP-B inhibits the cell viability of MM cells.U266 and RPMI8226 cells were incubated for 48 h with the indicated concentrations of NSP-B (0.5-40 µM).Cell viability assays were performed using CCK-8 as mentioned in Section 2. The graph displays the mean ± SD of three independent experiments.*p < .05,**p < .01,***p< .001.(b) NSP-B induces cell death in MM cell lines.U266 and RPMI8226 cells were treated with doses 1, 5, and 10 µM of NSP-B for 48 h.Then the cells were stained with live and dead reagent and visualized under a fluorescent microscope.(c) NSP-B-mediated apoptosis in MM cells.U266 and RPMI8226 cells were treated with NSP-B (1, 5, and 10 µM), followed by staining with fluorescein-conjugated Annexin-V/PI, and apoptotic cells were determined by flow cytometry.(d) Effect of NSP-B on cell cycle distribution.U266 and RPMI8226 cells were treated with NSP-B, and cell cycle fractions were determined with flow cytometry as described in the method and materials.(e) NSP-B-mediated phosphorylation of H2AX in MM cells.U266 and RPMI8226 cells were treated with NSP-B, and pH2AX level was determined by Western blot analysis using antibodies against p-H2AX and HSP60.Original Western blots, microscopic images and quantification graphs can be found at Files S1 and S2.CCK-8, Cell Counting Kit-8; MM, multiple myeloma; NSP-B, neosetophomone B; PI, propidium iodide.
,d) as well as lower doses S4, S7a,b).Notably, the global caspase inhibitor z-Vad-fmk was able to block NSP-B's effect on cleaved caspase-3 and -9 and PARP (Figure2e), indicating that NSP-B triggers caspasecascade signaling during apoptosis in MM cells.3.3 | NSP-B inhibits AKT activation, suppresses SKP2 and enhances p21, P27 expression in MM cells We investigated the effects of NSP-B on the AKT/PKB pathway in U266 and RPMI8226 cells by treating them with increasing concentrations of NSP-B (0, 1, 5, and 10 µM).The results showed that NSP-B caused a F I G U R E 2 NSP-B-induced mitochondrial signaling pathways in leukemic cell lines.(a) Effect of NSP-B on caspase-8/BID/Bcl2 and Bax expression in MM cell lines.U266 and RPMI8226 cells were treated with NSP-B as indicated, and 50 µg of protein was separated by SDS-PAGE and immunoblotted with antibodies against cleaved caspase-8, Bid, Bax, Bcl2, and HSP60 as indicated.(b) NSP-B-mediated loss of mitochondrial membrane potential in MM cells.U266 and RPMI8226 cells were treated with indicated doses (1, 5, 10 µM) of NSP-B for 48 h.Mitochondrial membrane potential was determined by flow cytometry as described in the materials and methods section and analyzed.(c) The NSP-B-induced the release of cytochrome c.For U266 same blot of (a) was stripped and probed against antibody cytochrome c.Same HSP60 was used for (a) and (c).RPMI8226 cells were treated with NSP-B, and proteins were separated on SDS-PAGE and immunoblotted with antibodies against cytochrome c and HSP60 (d) NSP-B mediates caspase activation in MM cells.U266 and RPMI8226 cells were treated with NSP-B, and proteins were separated on SDS-PAGE and immunoblotted with antibodies against caspase-9, cleaved caspase-9, caspase-3, cleaved caspase-3, PARP, and HSP60.(e) Effect of z-Vad-fmk on NSP-B induced apoptosis.U266 and RPMI8226 cells were pretreated with z-Vad-fmk for 1 h, followed by NSP-B treatment, and analyzed by western blot for antibodies against cleaved caspase-3, cleaved caspase 9, PARP, and HSP60.For U266 same blot of (e) was stripped and probed against antibodies PARP and HSP60.Original Western blot images and quantification graphs can be found at Files S1 and S2.MM, multiple myeloma; NSP-B, neosetophomone B; PARP, poly(ADP-ribose)polymerases; SDS-PAGE, sodium dodecyl-sulfate polyacrylamide gel electrophoresis.dephosphorylation of AKT at Ser473 and Thr308 without altering total AKT expression (Figure3a).Furthermore, NSP-B treatment led to a reduction in the levels of inhibitors of apoptosis protein (IAP), including XIAP, cIAP1, and cIAP2 in both U266 and RPMI8226 cells (Figure3a).In addition, NSP-B decreased SKP2 expression while increasing p21Cip1 and p27/kip1 levels(Figures 3b   and S7a,b), suggesting a mechanism for NSP-B-induced apoptosis through the inhibition of AKT and SKP2 signaling.To further confirm the involvement of AKT in NSP-B-induced apoptosis, we used siRNA to silence the AKT gene in U266 cells.AKTspecific siRNA was transfected into the cells using a Lonza nucleofector system, and the levels of AKT, SKP2, caspase-3, and HSP60 were measured using various antibodies.The results showed that knocking down the AKT gene reduced SKP2 expression and caspase-3 levels (Figure3c), suggesting that AKT inhibition inhibits cell growth and induces apoptosis in MM cells by downregulating SKP2.

3. 4 |
NSP-B causes the inhibition of cyclins and cyclin-dependent kinases (CDKs) in MM cells Our study aimed to examine the effects of NSP-B treatment on the expression of cyclins and CDKs in MM cells.Following 48 h of treatment with NSP-B, the expression of cyclin B1 and D1, as well as F I G U R E 3 NSP-B inhibits activated AKT/PKB, and induces downregulation of antiapoptotic proteins and SKP2 in MM cells.(a) NSP-B treatment caused the inactivation of AKT (both Ser473 and Thr308) and downregulated the expression of antiapoptotic proteins.U266 and RPMI8226 cells were treated with various doses of NSP-B.After cell lysis, equal amounts of proteins were separated by SDS-PAGE, transferred to the PVDF membrane, and immunoblotted with antibodies against XIAP, c-IAP1, c-IAP2 and HSP60 as indicated.(b) NSP-B treatment caused the downregulation of SKP2 and enhanced the expression levels of P27, P21, and ubiquitin.U266 and RPMI8226 cells were treated with various doses of NSP-B, and equal amounts of proteins were immunoblotted with antibodies against SKP2, p27, p21, ubiquitin, and HSP60 as indicated.(c) Gene silencing of AKT suppressed SKP2 expression.U266 cells were transfected with control (200 pM) and AKT siRNA (50, 100, and 200 pM) as indicated in Section 2. Immunoblot analysis of U266 cells transfected with control (200 pM) and AKT siRNA (50, 100, and 200 pM).Cells were lysed, and an equal amount of proteins for each sample were loaded onto the SDS-polyacrylamide gel.Membranes were blotted against AKT, SKP2, caspase 3, and HSP60.Original Western blot images and quantification graphs can be found in Files S1 and S2.MM, multiple myeloma; NSP-B, neosetophomone B; PVDF, polyvinylidene difluoride; SDS-PAGE, sodium dodecyl-sulfate polyacrylamide gel electrophoresis; siRNA, small interfering RNA.MM, multiple myeloma; NSP-B, neosetophomone B.F I G U R E 4 NSP-B inhibits the cyclins and CDKs in MM cells.(a) NSP-B treatment caused the downregulation of CDK-4 and -6 and cyclins B1 and D1.U266 and RPMI8226 cells were treated with various doses of NSP-B, and equal amounts of proteins were immunoblotted with antibodies against CDK-4, CDK-6, cyclin B1, cyclin D1, and HSP60 as indicated.Original Western blot images and quantification graphs can be found in Files S1 and S2.CDK, cyclin-dependent kinase; MM, multiple myeloma; NSP-B, neosetophomone B.

F
I G U R E 5 Combination of NSP-B and BTZ augmented the inhibition of cell viability and induces apoptosis in myeloma cells.(a) U266 and RPMI8226 were treated with NSP-B and BTZ alone and in combination for 48 h.After incubation, the cells were stained with live and dead reagent and visualized under a fluorescent microscope.(b) U266 and RPMI8226 were treated with 0.5 μM of NSP-B and 10 nM BTZ for U266 and 2.5 nM BTZ for RPMI8226 alone and in combination.Cells were lysed and separated using SDS-PAGE, transferred to a PVDF membrane, and immunoblotted with antibodies such as caspase 3, PARP, and HSP60.Original Western blots, microscopic images, and quantification graphs can be found at Files S1 and S2.BTZ, bortezomib; NSP-B, neosetophomone B; PARP, poly(ADP-ribose)polymerases; PVDF, polyvinylidene difluoride; SDS-PAGE, sodium dodecyl-sulfate polyacrylamide gel electrophoresis.
versatility and adaptability of existing chemotherapeutic drugs when paired with targeted inhibitors, opening novel therapeutic avenues in our relentless pursuit of effective cancer treatments.In the present study, we evaluated the potential for combination therapy with subtoxic concentrations of NSP-B and BTZ, an FDA-approved treatment for MM, to elicit a synergistic or additive effect that selectively kills tumor cells without harming healthy cells.Our results demonstrated that the combination of NSP-B and BTZ reduced the viability of U266 and RPMI8226 cells and induced apoptosis more effectively than either drug alone.NSP-B sensitized MM cells to BTZ, significantly reducing cell proliferation and enhancing cell death.These findings suggest that the use of sub-toxic doses of NSP-B in combination with BTZ may offer a promising strategy for improving the therapeutic outcomes of MM treatment.5 | CONCLUSION Our findings indicate that NSP-B effectively inhibits growth and induces apoptosis in MM cell lines (U266 and RPMI8226).NSP-B treatment reduced the activation of AKT, decreased the IAPs, and modulated the expression of SKP2, p21Kip1, and p27Cip1.Moreover, we observed a higher Bax/BCL2 ratio, loss of MMP, and cytochrome c release into the cytoplasm, thereby initiating caspasedependent apoptosis (Figure 6).NSP-B also enhanced the cytotoxic F I G U R E 6 Schematic digram showing possible mechanisms of NSP-B-mediated anticancer activity in MM cells.MM, multiple myeloma; NSP-B, neosetophomone B. in cotreatment.These observations strongly suggest the potential of NSP-B as a potent anticancer agent against MM.