Thymol-loaded liposomes effectively induced apoptosis and decreased EGFR expression in colorectal cancer cells.

BACKGROUND: Colorectal cancer (CRC) is one of the most common and deadly cancers worldwide. Different factors, such as environmental and genetic factors and lifestyle, affect it. Owing to the presence of phenolic, alkaloid, antioxidant, and terpenoid compounds, herbal compounds can be effective in the treatment of various cancers. Thymol is a natural monoterpene phenol that is abundant in some plants and exerts several biological effects. The aim of this study was to investigate the apoptotic, anti-proliferative effect and EGFR gene expression under the influence of thymol-loaded nanoliposome in SW84 and SW111 cell lines derived from colorectal cancer. MATERIALS AND METHODS: The lipid thin-film hydration method was used to synthesize thymol-loaded liposomes, and their characterization was performed using TEM, DLS, and HPLC analyses. SW84 and SW1111 cells were treated with thymol- and thymol-loaded liposomes at different doses, the inhibition of cell proliferation was evaluated using an MTT assay, the rate of apoptosis induction was assessed using flow cytometry, and EGFR gene expression was measured using real-time PCR. RESULTS: The nanoparticles produced were spherical, uniform, and 200 ± 10 nm in size. HPLC analysis showed that approximately 98% thymol was loaded into the nanoliposome. The results of the MTT assay showed that thymol and thymol-nanoliposomes decreased the proliferation of SW84 and SW1111 cells in a concentration-dependent manner. The IC50 of thymol and thymol-nanoliposomes were 18 and 14.2 µg/ml for the SW48 cell line (P = 0.04) and 10.5 and 6.4 µg/ml for the SW1116 cell line (P = 0.001). Thymol-nanoliposomes significantly inhibited the proliferation of cancer cells compared to free thymol. Flow cytometry showed an increase in the percentage of apoptotic cells, especially in the thymol-nanoliposome group in the treated cells. Real-time PCR results also showed that thymol and thymol-nanoliposome both caused a decrease in the expression of EGFR genes in both cell lines, but this effect of decreasing gene expression was significantly higher in the thymol-nanoliposome group. CONCLUSIONS: Our results showed that thymol-nanoliposomes reduced proliferation, increased apoptosis, and decreased EGFR expression in colorectal cancer-derived cell lines.

Quercetin-loaded Liposomes Effectively Induced Apoptosis and Decreased the Epidermal Growth Factor Receptor Expression in Colorectal Cancer Cells: An In Vitro Study.

Background: Quercetin is a flavonoid having anti-cancer properties; however, it has low stability, insufficient bioavailability, and poor solubility. This study aimed to load quercetin on nanoliposomes to enhance its efficiency against SW48 colorectal cancer cells. The cytotoxicity of free-quercetin and quercetin-loaded nanoliposomes on the expression of the epidermal growth factor receptor (EGER) gene was investigated. Methods: This present in vitro study was conducted at Yasuj University of Medical Sciences (Yasuj, Iran) in 2021. In this in vitro study, the lipid thin-film hydration method was used to synthesize quercetin-loaded liposomes. Additionally, high-performance liquid chromatography (HPLC) analyses, dynamic light scattering (DLS), and transmission electron microscopy (TEM) investigations were used to characterize nanomaterials. Following that, MTT, flow cytometry, and real-time PCR were used to investigate the cytotoxicity of quercetin-loaded liposomes on the colorectal cancer cells SW48 cell line, the incidence of apoptosis, and the expression of the EGFR gene in these cells. Statistical analysis was performed using the SPSS (version 26.0), and the graphs were created with the GraphPad Prism version 8.4.3. P<0.05 was considered statistically significant. Results: The nanoparticles were spherical, homogenous, and 150±10 nm in size. According to HPLC, Quercetin had a 98% loading capacity. Although both free quercetin and quercetin-loaded liposomes indicated significant cytotoxicity against cancer cells (P˂0.001), the combined form was significantly more active (P=0.008). 50 µg/mL of this compound reduced the viability of SW48 cells by more than 80% (IC50 10.65 µg/mL), while the viability of cells treated with free quercetin was only 66% (IC50 18.74 µg/mL). The apoptosis was nearly doubled in the cells treated with quercetin-loaded nanoliposomes compared to free quercetin (54.8% versus 27.6%). EGFR gene expression, on the other hand, was significantly lower in cells treated with quercetin-loaded liposomes than the quercetin alone (P=0.006). Conclusion: When combined with nanoliposomes, quercetin had greater anti-proliferative, apoptotic, and anti-EGFR expression than free quercetin.

Safflower seed oil, a rich source of linoleic acid, stimulates hypothalamic neurogenesis in vivo.

Adult neurogenesis has been reported in the hypothalamus, subventricular zone and subgranular zone in the hippocamp. Recent studies indicated that new cells in the hypothalamus are affected by diet. We previously showed beneficial effects of safflower seed oil (SSO), a rich source of linoleic acid (LA; 74%), on proliferation and differentiation of neural stem cells (NSCs) in vitro. In this study, the effect of SSO on hypothalamic neurogenesis was investigated in vivo, in comparison to synthetic LA. Adult mice were treated with SSO (400 mg/kg) and pure synthetic LA (300 mg/kg), at similar concentrations of LA, for 8 weeks and then hypothalamic NSCs were cultured and subsequently used for Neurosphere-forming assay. In addition, serum levels of brain-derived neurotrophic factor (BNDF) were measured using enzyme-linked immunosorbent assay. Administration of SSO for 8 weeks in adult mice promoted the proliferation of NSCs isolated from SSO-treated mice. Immunofluorescence staining of the hypothalamus showed that the frequency of astrocytes (glial fibrillary acidic protein+ cells) are not affected by LA or SSO. However, the frequency of immature (doublecortin+ cells) and mature (neuronal nuclei+ cells) neurons significantly increased in LA- and SSO-treated mice, compared to vehicle. Furthermore, both LA and SSO caused a significant increase in the serum levels of BDNF. Importantly, SSO acted more potently than LA in all experiments. The presence of other fatty acids in SSO, such as oleic acid and palmitic acid, suggests that they could be responsible for SSO positive effect on hypothalamic proliferation and neurogenesis, compared to synthetic LA at similar concentrations.

Bispecific antibodies in colorectal cancer therapy: recent insights and emerging concepts.

Colorectal cancer (CRC) is identified as a life-threatening malignancy. Despite several efforts and proceedings available for CRC therapy, it is still a health concern. Among a vast array of novel therapeutic procedures, employing bispecific antibodies (BsAbs) is currently considered to be a promising approach for cancer therapy. BsAbs, as a large family of molecules designed to realize two distinct epitopes or antigens, can be beneficial microgadgets to target the tumor-associated antigen pairs. On the other hand, applying the immune system's capabilities to attack malignant cells has been proven as a tremendous development in cancer therapeutic projects. The current study has attempted to overview some of the approved BsAbs in CRC therapy and those under clinical trials. For this purpose, reputable scientific search engines and databases, such as PubMed, ScienceDirect, Google Scholar, Scopus, etc., were explored using the keywords 'bispecific antibodies', 'colorectal cancer', 'immunotherapy' and 'tumor markers'.

The emerging role of microRNA in regulating the mTOR signaling pathway in immune and inflammatory responses.

The mechanistic/mammalian target of rapamycin (mTOR) is considered to be an atypical protein kinase that plays a critical role in integrating different cellular and environmental inputs in the form of growth factors, nutrients and energy and, subsequently, in regulating different cellular events, including cell metabolism, survival, homeostasis, growth and cellular differentiation. Immunologically, mTOR is a critical regulator of immune function through integrating numerous signals from the immune microenvironment, which coordinates the functions of immune cells and T cell fate decisions. The crucial role of mTOR in immune responses has been lately even more appreciated. MicroRNAs (miRNAs) are endogenous, small, noncoding single-stranded RNAs that act as molecular regulators involved in multiple processes during immune cells development, homeostasis, activation and effector polarization. Several studies have recently indicated that a range of miRNAs are involved in regulating the phosphoinositide 3-kinase/protein kinase B/mTOR (PI3K/AKT/mTOR) signaling pathway by targeting multiple components of this signaling pathway and modulating the expression and function of these targets. Current evidence has revealed the interplay between miRNAs and the mTOR pathway circuits in various immune cell types. The expression of individual miRNA can affect the function of mTOR signaling to determine the cell fate decisions in immune responses through coordinating immune signaling and cell metabolism. Dysregulation of the mTOR pathway/miRNAs crosstalk has been reported in cancers and various immune-related diseases. Thus, expression profiles of dysregulated miRNAs could influence the mTOR pathway, resulting in the promotion of aberrant immunity. This review summarizes the latest information regarding the reciprocal role of the mTOR signaling pathway and miRNAs in orchestrating immune responses.

Codelivery of HIF-1α siRNA and Dinaciclib by Carboxylated Graphene Oxide-Trimethyl Chitosan-Hyaluronate Nanoparticles Significantly Suppresses Cancer Cell Progression.

PURPOSE: Hypoxia-inducible factor (HIF) is one of the critical components of the tumor microenvironment that is involved in tumor development. HIF-1α functionally and physically interacts with CDK1, 2, and 5 and stimulates the cell cycle progression and Cyclin-Dependent Kinase (CDK) expression. Therefore, hypoxic tumor microenvironment and CDK overexpression lead to increased cell cycle progression and tumor expansion. Therefore, we decided to suppress cancer cell expansion by blocking HIF-1α and CDK molecules. METHODS: In the present study, we used the carboxylated graphene oxide (CGO) conjugated with trimethyl chitosan (TMC) and hyaluronate (HA) nanoparticles (NPs) loaded with HIF-1α-siRNA and Dinaciclib, the CDK inhibitor, for silencing HIF-1α and blockade of CDKs in CD44-expressing cancer cells and evaluated the impact of combination therapy on proliferation, metastasis, apoptosis, and tumor growth. RESULTS: The results indicated that the manufactured NPs had conceivable physicochemical properties, high cellular uptake, and low toxicity. Moreover, combination therapy of cancer cells using CGO-TMC-HA NPs loaded with HIF-1α siRNA and Dinaciclib (SCH 727965) significantly suppressed the CDKs/HIF-1α and consequently, decreased the proliferation, migration, angiogenesis, and colony formation in tumor cells. CONCLUSIONS: These results indicate the ability of CGO-TMC-HA NPs for dual drug/gene delivery in cancer treatment. Furthermore, the simultaneous inhibition of CDKs/HIF-1α can be considered as a novel anti-cancer treatment strategy; however, further research is needed to confirm this treatment in vivo. Graphical Abstract The suppression of HIF-1α and CDKs inhibits cancer growth. HIF-1α is overexpressed by the cells present in the tumor microenvironment. The hypoxic environment elevates mitochondrial ROS production and increases p38 MAP kinase, JAK/STAT, ERK, JNK, and Akt/PI3K signaling, resulting in cyclin accumulation and aberrant cell cycle progression. Furthermore, the overexpression of HIF-1α/CDK results in increased expression of genes such as BCL2, Bcl-xl, Ki-67, TGFß, VEGF, FGF, MMP2, MMP9, and, HIF-1α and consequently raise the survival, proliferation, angiogenesis, metastasis, and invasion of tumor cells. In conclusion, HIF-1α-siRNA/Dinaciclib-loaded CGO-TMC-HA NPs can inhibit the tumor expansion by blockage of CDKs and HIF-1α (JAK: Janus kinase, STAT: Signal transducer and activator of transcription, MAPK: mitogen-activated protein kinase, ERK: extracellular signal-regulated kinase, JNK: c-Jun N-terminal kinase, PI3K: phosphatidylinositol 3-kinase).

Adenosine and adenosine receptors in colorectal cancer.

CD39 (nucleoside triphosphate diphosphohydrolase) and Ecto-5-nucleotidase (CD73) have been recognized as important factors mediating various pathological and physiological responses in the tumor microenvironment. Elevated expression of CD73 and CD39 is correlated with the over-production of adenosine in the tumor region. This increase is associated with an immunosuppressive state in the tumor site that enhances various tumor hallmarks such as metastasis, angiogenesis, and cell proliferation. Adenosine promotes these behaviors through interaction with four adenosine receptors, including A3R, A2BR, A2AR, and A1R. Signaling of these receptors reduces the function of immune effector cells and enhances the expansion and function of tumor-associated immune cells. Several studies have been shown the important role of adenosine/CD73/CD39/ARs axis in the immunopathogenesis of colorectal cancer. These findings imply that components of this axis can be considered as a worthy target for colorectal cancer immunotherapy. In this review, we summarized the role of CD73/CD39/adenosine/ARs in the immunopathogenesis of colorectal cancer.

Inhibition of CD73 using folate targeted nanoparticles carrying anti-CD73 siRNA potentiates anticancer efficacy of Dinaciclib.

Conventional therapeutic methods against cancer, including chemotherapy, radiotherapy, surgery, and combination therapy, have exhibited different toxicity levels due to their unspecific mechanism of action. To overcome the challenges facing conventional cancer therapies, newly developed methods are being investigated. Significant levels of specificity, remarkable accumulation at the tumor site, limited side effects, and minimal off-target effects enable the newly synthesized nanoparticles (NPs) to become the preferred drug delivery method in anticancer therapeutic approaches. According to the literature, CD73 has a pivotal role in cancer progression and resistance to chemotherapy and radiotherapy. Therefore, CD73 has attracted considerable attention among scientists to target this molecule. Accordingly, FDA approved CDK inhibitors such as Dinaciclib that blocks CDK1, 2, 5, and 9, and exhibits significant anticancer activity. So in this study, we intended to simultaneously suppress CD73 and CDKs in cancer cells by using the folic acid (FA)-conjugated chitosan-lactate (CL) NPs loaded with anti-CD73 siRNA and Dinaciclib to control tumor progression and metastasis. The results showed that NPs could effectively transfect cancer cells in a FA receptor-dependent manner leading to suppression of proliferation, survival, migration, and metastatic potential. Moreover, the treatment of tumor-bearing mice with this combination strategy robustly inhibited tumor growth and enhanced survival time in mice. These findings imply the high potential of FA-CL NPs loaded with anti-CD73 siRNA and Dinaciclib for use in cancer treatment shortly.

Concomitant blockade of A2AR and CTLA-4 by siRNA-loaded polyethylene glycol-chitosan-alginate nanoparticles synergistically enhances antitumor T-cell responses.

Inhibitory immune checkpoint (ICP) molecules are important immunosuppressive factors in a tumor microenvironment (TME). They can robustly suppress T-cell-mediated antitumor immune responses leading to cancer progression. Among the checkpoint molecules, cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) is one of the critical inhibitors of anticancer T-cell responses. Besides, the expression of adenosine receptor (A2AR) on tumor-infiltrating T cells potently reduces their function. We hypothesized that concomitant silencing of these molecules in T cells might lead to enhanced antitumor responses. To examine this assumption, we purified T cells from the tumor, spleen, and local lymph nodes of CT26 colon cancer-bearing mice and suppressed the expression of A2AR and CTLA-4 using the small interfering RNA (siRNA)-loaded polyethylene glycol-chitosan-alginate (PCA) nanoparticles. The appropriate physicochemical properties of the produced nanoparticles (NPs; size of 72 nm, polydispersive index [PDI] < 0.2, and zeta potential of 11 mV) resulted in their high efficiency in transfection and suppression of target gene expression. Following the silencing of checkpoint molecules, various T-cell functions, including proliferation, apoptosis, cytokine secretion, differentiation, and cytotoxicity were analyzed, ex vivo. The results showed that the generated nanoparticles had optimal physicochemical characteristics and significantly suppressed the expression of target molecules in T cells. Moreover, a concomitant blockade of A2AR and CTLA-4 in T cells could synergistically enhance antitumor responses through the downregulation of PKA, SHP2, and PP2Aα signaling pathways. Therefore, this combination therapy can be considered as a novel promising anticancer therapeutic strategy, which should be further investigated in subsequent studies.

Silencing of HIF-1α/CD73 axis by siRNA-loaded TAT-chitosan-spion nanoparticles robustly blocks cancer cell progression.

Induction of Hypoxia Inducible Factor (HIF) as a direct consequence of oxygen deficiency in tumor tissues is a potent stimulus of CD73 (ecto-5'-nucleotidase) expression. Hypoxic environment and CD73 overexpression are associated with altered metabolism, elevated cancer cell proliferation, and tumor vascularization. Herein, a delivery system was developed for silencing CD73 and HIF-1α gene using siRNA-loaded Superparamagnetic iron oxide (SPION) nanocarriers for cancer treatment. SPIONs were encapsulated with thiolated chitosan (TC) and trimethyl chitosan (TMC) for improving their stabilization and functionalization. The nanoparticles (NPs) were about 133 nm in size, spherical, and non-toxic, and the addition of TAT peptide (derived from HIV-1 TAT protein) to TMC-TC-SPIONs significantly increased their cellular uptake by cancer cells. The produced NPs could efficiently accumulate in the tumor site, indicating their stability and targeting ability in reaching the tumor region. TAT-conjugated TMC-TC-SPIONs containing siRNAs could significantly reduce the HIF-1α and CD73 expression levels in cancer cells. Following transfection, cancer cells showed a significant reduction in migration and proliferation. Moreover, siRNA-loaded NPs could effectively reduce tumor growth and angiogenesis, as investigated by the chick chorioallantoic membrane (CAM) assay. This study suggested that TAT-TMC-TC-SPIONs can be potential nanocarrier for gene transfection in cancer therapy. Moreover, the co-silencing of CD73 and HIF-1α can be assumed as a novel anti-cancer treatment strategy with high tumor suppression potential.

Blockade of CTLA-4 increases anti-tumor response inducing potential of dendritic cell vaccine.

The immunosuppressive state of the tumor microenvironment diminishes the efficacy of dendritic cell (DC)-based cancer immunotherapy. Inhibitory immune checkpoint molecules expressed on tumor-infiltrating T lymphocytes, such as cytotoxic T-lymphocyte antigen 4 (CTLA-4) molecules are one of the main barriers in priming T cells by DCs. Therefore, it seems that blockade of such molecules facilitates the T cells activation by the DC vaccine. In this study, we intended to suppress the expression of CTLA-4 molecule on tumor-infiltrating T cells by siRNA-loaded chitosan-lactate (CL) nanoparticles to facilitate priming anti- tumor T cells by tumor lysate-loaded DC vaccine. Nanoparticles (NPs) have also provided an opportunity for specific drug delivery into the tumor site. CL NPs exhibited favorable physicochemical characteristics (size about 75 nm, polydispersive index<0.2, and a zeta potential about 14), which were associated with a high transfection rate and low toxicity. Moreover, the administration of anti-CTLA-4 siRNA-loaded NPs into CT26 and 4 T1 tumor -bearing mice led to the downregulation of CTLA-4 on tumor -infiltrating T cells, which was associated with tumor regression and increased mice survival. Moreover, while the treatment of tumor -bearing mice with DC vaccine had mild therapeutic outcomes, its combination with siRNA-loaded NPs may exhibit synergistic anti- tumor effects. This possible synergistic ameliorating effect is achieved through the reduction of immunosuppressive cells, the improved cytotoxicity of T lymphocytes, decreased inhibitory and increased inflammatory cytokines, and reduced angiogenesis and metastasis processes. These results indicate that the silencing of CTLA-4 can potentiate the T cell priming capacity of the DC vaccine, proposing a practical anti-cancer therapeutic approach.

Silencing adenosine A2a receptor enhances dendritic cell-based cancer immunotherapy.

Overexpression of adenosine in the tumor region leads to suppression of various immune cells, particularly T cells through ligation with adenosine 2a receptor (A2aR). In this study, we intended to increase the efficacy of tumor lysate-loaded DC vaccine by silencing the expression of A2aR on T cells through the application of A2aR-specific siRNA-loaded PEG-chitosan-lactate (PCL) nanoparticles (NPs) in the 4T1 breast tumor-bearing mice. Combination therapy by DC vaccine and siRNA-loaded NPs markedly induced tumor regression and increased survival time of mice. These ameliorative effects were partly via downregulation of immunosuppressive cells, increased function of cytotoxic T lymphocytes, and induction of immune-stimulatory cytokines. Moreover, combination therapy could markedly suppress angiogenesis and metastasis processes. These results imply the efficacy of novel combination therapy for the treatment of breast cancer by using A2aR siRNA-loaded NPs and DC vaccine which can be translated into the initial phase of clinical trials in the near future.

Coinhibition of S1PR1 and GP130 by siRNA-loaded alginate-conjugated trimethyl chitosan nanoparticles robustly blocks development of cancer cells.

There is an interconnected network between S1P/sphingosine-1-phosphate receptor 1 (S1PR1), IL-6/glycoprotein 130 (GP130), and signal transducer and activator of transcription 3 (STAT3) signaling pathways in the tumor microenvironment, which leads to cancer progression. S1P/S1PR1 and IL-6/GP130 signaling pathways phosphorylate and activate STAT3, and it then induces the expression of S1PR1 and interleukin-6 (IL-6) in a positive feedback loop leading to cancer progression. We hypothesized that blockade of this amplification loop can suppress the growth and development of cancer cells. Therefore, we silenced STAT3 upstream molecules including the S1PR1 and GP130 molecules in cancer cells using small interfering RNA (siRNA)-loaded alginate-conjugated trimethyl chitosan (ATMC) nanoparticles (NPs). The generated NPs had competent properties including the appropriate size, zeta potential, polydispersity index, morphology, high uptake of siRNA, high rate of capacity, high stability, and low toxicity. We evaluated the effects of siRNA loaded ATMC NPs on tumor hallmarks of three murine-derived cancer cell lines, including 4T1 (breast cancer), B16-F10 (melanoma), and CT26 (colon cancer). The results confirmed the tumor-suppressive effects of combinational targeting of S1PR1 and GP130. Moreover, combination therapy could potently suppress tumor growth as assessed by the chick chorioallantoic membrane assay. In this study, we targeted this positive feedback loop for the first time and applied this novel combination therapy, which provides a promising approach for cancer treatment. The development of a potent nanocarrier system with ATMC for this combination was also another aspect of this study, which should be further investigated in cancer animal models in further studies.

Tumor associated macrophages in the molecular pathogenesis of ovarian cancer.

The tumor microenvironment is a critical factor that enhances cancer progression, drug resistance, and failure of therapeutic approaches. Several cellular and non-cellular factors are involved in cancer promotion. Among the several cell populations in the tumor microenvironment, macrophages, as one of the most abundant innate immune cells within the tumor milieu, have attracted extensive attention among several researchers because of their critical role in innate pathophysiology of multiple disorders, as well as ovarian cancer. High plasticity and consequent high ability to adapt to environmental alternations by adjusting their cellular metabolism and immunological phenotype is the notable characteristic of macrophages. Therefore, the critical function of tumor-associated macrophages in ovarian cancer is highlighted in the growing body of recent studies. In this article, we will comprehensively focus on significant impacts of the macrophages on ovarian cancer progression, by discussing the role of macrophages as one of the fundamental immune cells present in tumor milieu, in metabolic reprogramming of transformed cells, and involvement of these cells in the ovarian cancer initiation, progression, invasion, and angiogenesis. Moreover, we will summarise recent studies evaluating the effects of targeting macrophages in ovarian cancer.

Nanomedicine for improvement of dendritic cell-based cancer immunotherapy.

Dendritic cell (DC)-based cancer immunotherapy has shown impressive outcomes, including the development of the first FDA-approved anti-cancer vaccine. However, the clinical application of DC-based cancer immunotherapy is associated with various challenges. Promising novel tools for the administration of cancer vaccines has emerged from recent developments in nanoscale biomaterials. One current strategy to enhance targeted drug delivery, while minimizing drug-related toxicities, is the use of nanoparticles (NPs). These can be utilized for antigen delivery into DCs, which have been shown to provide potent T cell-stimulating effects. Therefore, NP delivery represents one promising approach for creating an effective and stable immune response without toxic side effects. The current review surveys cancer immunotherapy with particular attention toward NP-based delivery methods that target DCs.

Codelivery of STAT3 siRNA and BV6 by carboxymethyl dextran trimethyl chitosan nanoparticles suppresses cancer cell progression.

High expression of inhibitor of apoptosis (IAP) molecules in cancer cells promotes cancer cell chemoresistance. Use of BV6, a well-known IAP inhibitor, along with inhibition of signal transducer and activator of transcription 3 (STAT3), which is an important factor in the survival of tumor cells, and NIK as a mediator of BV6 unpredicted side effects, can induce effective apoptosis in tumor cells. The present study has investigated the combination therapy of cancer cells using Carboxymethyl Dextran-conjugated trimethyl chitosan (TMC-CMD) nanoparticles (NPs) loaded with NIK/STAT3-specific siRNA and BV6 to synergistically induce apoptosis in the breast, colorectal and melanoma cancer cell lines. Our results showed that in addition to enhanced pro-apoptotic effects, this combination therapy reduced proliferation, cell migration, colony formation, and angiogenesis, along with expression of factors including IL-10 and HIF in tumor cells. The results indicate the potential of this combination therapy for further investigation in animal models of cancer.