Gulp1 deficiency augments bone mass in male mice by affecting osteoclasts due to elevated 17ß-estradiol levels.

The engulfment adaptor phosphotyrosine-binding domain containing 1 (GULP1) is an adaptor protein involved in the engulfment of apoptotic cells via phagocytosis. Gulp1 was first found to promote the phagocytosis of apoptotic cells by macrophages, and its role in various tissues, including neurons and ovaries, has been well studied. However, the expression and function of GULP1 in bone tissue are poorly understood. Consequently, to determine whether GULP1 plays a role in the regulation of bone remodeling in vitro and in vivo, we generated Gulp1 knockout (KO) mice. Gulp1 was expressed in bone tissue, mainly in osteoblasts, while its expression is very low in osteoclasts. Microcomputed tomography and histomorphometry analysis in 8-week-old male Gulp1 KO mice revealed a high bone mass in comparison with male wild-type (WT) mice. This was a result of decreased osteoclast differentiation and function in vivo and in vitro as confirmed by a reduced actin ring and microtubule formation in osteoclasts. Gas chromatography-mass spectrometry analysis further showed that both 17ß-estradiol (E2) and 2-hydroxyestradiol levels, and the E2/testosterone metabolic ratio, reflecting aromatase activity, were also higher in the bone marrow of male Gulp1 KO mice than in male WT mice. Consistent with mass spectrometry analysis, aromatase enzymatic activity was significantly higher in the bone marrow of male Gulp1 KO mice. Altogether, our results suggest that GULP1 deficiency decreases the differentiation and function of osteoclasts themselves and increases sex steroid hormone-mediated inhibition of osteoclast differentiation and function, rather than affecting osteoblasts, resulting in a high bone mass in male mice. To the best of our knowledge, this is the first study to explore the direct and indirect roles of GULP1 in bone remodeling, providing new insights into its regulation.

Statin-mediated inhibition of RAS prenylation activates ER stress to enhance the immunogenicity of KRAS mutant cancer.

BACKGROUND: Statins preferentially promote tumor-specific apoptosis by depleting isoprenoid such as farnesyl pyrophosphate and geranylgeranyl pyrophosphate. However, statins have not yet been approved for clinical cancer treatment due, in part, to poor understanding of molecular determinants on statin sensitivity. Here, we investigated the potential of statins to elicit enhanced immunogenicity of KRAS-mutant (KRASmut) tumors. METHODS: The immunogenicity of treated cancer cells was determined by western blot, flow cytometry and confocal microscopy. The immunotherapeutic efficacy of mono or combination therapy using statin was assessed in KRASmut tumor models, including syngeneic colorectal cancer and genetically engineered lung and pancreatic tumors. Using NanoString analysis, we analyzed how statin influenced the gene signatures associated with the antigen presentation of dendritic cells in vivo and evaluated whether statin could induce CD8+ T-cell immunity. Multiplex immunohistochemistry was performed to better understand the complicated tumor-immune microenvironment. RESULTS: Statin-mediated inhibition of KRAS prenylation provoked severe endoplasmic reticulum (ER) stress by attenuating the anti-ER stress effect of KRAS mutation, thereby resulting in the immunogenic cell death (ICD) of KRASmut cancer cells. Moreover, statin-mediated ICD enhanced the cross-priming ability of dendritic cells, thereby provoking CD8+ T-cell immune responses against KRASmut tumors. Combination therapy using statin and oxaliplatin, an ICD inducer, significantly enhanced the immunogenicity of KRASmut tumors and promoted tumor-specific immunity in syngeneic and genetically engineered KRASmut tumor models. Along with immune-checkpoint inhibitors, the abovementioned combination therapy overcame resistance to PD-1 blockade therapies, improving the survival rate of KRASmut tumor models. CONCLUSIONS: Our findings suggest that KRAS mutation could be a molecular target for statins to elicit potent tumor-specific immunity.

Nkx-2.5 Regulates MDR1 Expression via Its Upstream Promoter in Breast Cancer Cells.

BACKGROUND: Increased expression of MDR1 gene is one of the major mechanisms responsible for multidrug resistance in cancer cells. Two alternative promoters, upstream and downstream, are responsible for transcription of MDR1 gene in the human. However, the molecular mechanism regarding the transactivation of MDR1 upstream promoter (USP) has not been determined. METHODS: Dual-luciferase reporter gene assays were used to assess the effect of Nkx-2.5 on MDR1 USP activity using reporter plasmids for human MDR1 USP and its mutants. MDR1 mRNA level was examined by quantitative real-time PCR. The direct binding of Nkx-2.5 to the USP of MDR1 was evaluated by promoter enzyme immunoassays and chromatin immunoprecipitation assays. RESULTS: Nkx-2.5 significantly stimulates the transactivation of MDR1 USP and increases MDR1 mRNA expression in MCF7 breast cancer cells. Reporter gene assays with deleted MDR1 USPs showed that the Nkx-2.5-binding site is located between positions -71 and +12. Mutation of the Nkx-2.5-binding site at nucleotide +4 to +10 markedly reduced the Nkx-2.5-mediated activation of MDR1 USP activity. A promoter binding immunoassay and a chromatin immunoprecipitation assay revealed that Nkx-2.5 binds directly to the region +4/+10 of human MDR1 USP. CONCLUSION: The results in the present study show Nkx-2.5 is a positive regulator for the transactivation of MDR1 USP in MCF7 breast cancer cells. Our findings will help elucidate the regulatory mechanism responsible for the multidrug resistant cancer phenotype.

Ablation of Stabilin-1 Enhances Bone-Resorbing Activity in Osteoclasts In Vitro.

Stabilin-1 is a transmembrane receptor that regulates molecule recycling and cell homeostasis by controlling the intracellular trafficking and participates in cell-cell adhesion and transmigration. Stabilin-1 expression is observed in various organs, including bones; however, its function and regulatory mechanisms in the bone remain unclear. In this study, we evaluated the physiological function of stabilin-1 in bone cells and tissue using a stabilin-1 knockout (Stab1 KO) mouse model. In wild-type (WT) mice, stabilin-1 was expressed in osteoblasts and osteoclasts, and its expression was maintained during osteoblast differentiation but significantly decreased after osteoclast differentiation. There was no difference in osteoblast differentiation and function, or the expression of osteoblast differentiation markers between mesenchymal stem cells isolated from Stab1 KO and WT mice. However, osteoclast differentiation marker levels demonstrated a non-significant increase and bone-resorbing activity was significantly increased in vitro in RANKL-induced osteoclasts from Stab1-deficient bone marrow macrophages (BMMs) compared with those of WT BMMs. Microcomputed tomography showed a negligible difference between WT and Stab1 KO mice in bone volume and trabecular thickness and number. Moreover, no in vivo functional defect in bone formation by osteoblasts was observed in the Stab1 KO mice. The osteoclast surface and number showed an increased tendency in Stab1 KO mice compared to WT mice in vivo, but this difference was not statistically significant. Overall, these results indicate that Stab1 does not play an essential role in in vivo bone development and bone cell function, but it does affect in vitro osteoclast maturation and function for bone resorption.

Harnessing immune checkpoints in myeloid lineage cells for cancer immunotherapy.

Myeloid lineage immune cells, such as macrophages and dendritic cells, play important roles in the induction of antitumor immunity during the initial stage of the cancer-immunity cycle, eliciting antitumor adaptive immunity by phagocytosing cancer cells and processing cancer-specific antigens, and then presenting these antigens to T cells. During this process, cancer cell phagocytosis can be prevented by inhibitory signals, and the signaling cascades that elicit immune responses against cancer antigens can be inhibited by immunosuppressive myeloid cells in the tumor microenvironment. A number of therapeutic strategies for enhancing cancer cell phagocytosis and promoting antitumor immunity by targeting myeloid lineage cells have recently been developed. Here, we discuss recent advances in cancer immunotherapy that involve the targeting of myeloid lineage immune cells to induce effective antitumor immunity.

C1q and TNF related protein 1 regulates expression of inflammatory genes in vascular smooth muscle cells.

BACKGROUND: C1q and TNF related protein 1 (C1QTNF1) is known to be associated with coronary artery diseases. However, the molecular function of C1QTNF1 on the vascular smooth muscles remains to be investigated. OBJECTIVE: This study was therefore undertaken to investigate the effect of C1QTNF1 on gene expression of human smooth muscle cells and to reveal potential molecular mechanisms mediated by C1QTNF1. METHODS: Vascular smooth muscle cells were incubated with recombinant C1QTNF1 for 16 h, followed by determining any change in mRNA expressions by Affymetrix genechip. Gene ontology (GO), KEGG pathway, and protein-protein interaction (PPI) network were analyzed in differentially expressed genes. In addition, validation of microarray data was performed using quantitative real-time PCR. RESULTS: The mRNA expressions of annotated 74 genes were significantly altered after incubation with recombinant C1QTNF1; 41 genes were up-regulated and 33 down-regulated. The differentially expressed genes were enriched in biological processes and KEGG pathways associated with inflammatory responses. In the PPI network analysis, IL-6, CCL2, and ICAM1 were identified as potential key genes with relatively high degree. The cluster analysis in the PPI network identified a significant module composed of upregulated genes, such as IL-6, CCL2, NFKBIA, SOD2, and ICAM1. The quantitative real-time PCR results of potential key genes were consistent with microarray data. CONCLUSION: The results in the present study provide insights on the effects of C1QTNF1 on gene expression of smooth muscle cells. We believe our findings will help to elucidate the molecular mechanisms regarding the functions of C1QTNF1 on smooth muscle cells in inflammatory diseases.

Macrophagic Stabilin-1 Restored Disruption of Vascular Integrity Caused by Sepsis.

Sepsis develops because of overwhelming inflammatory responses to bacterial infection, and disrupts vascular integrity. Stabilin-1 (STAB-1) is a phagocytic receptor, which mediates efferocytosis in a phosphatidylserine (PS)-dependent manner. STAB-1 is expected to play important roles in efferocytosis during sepsis. Here, we determined the role of STAB-1 in maintaining and restoring vascular integrity. Macrophages and vascular endothelial cells were used to assess the effect of STAB-1 on survival rate, phagocytic activity, vascular permeability and transendothelial migration (TEM). Additionally, we investigated whether the high-mobility group box 1 (HMGB1)-receptor for advanced glycated end products complex interfered with the binding of Stab1 to PS. Mortality rate was higher in the Stab1-knockout mice than in the wild-type mice, and STAB-1 deficiency was related to reduced macrophage-mediated efferocytosis and the disruption of vascular integrity, which increased vascular permeability, and enhanced TEM. STAB-1 deficiency promoted lung injury, and elevated the expression of sepsis markers. The exogenous application of the anti-HMGB1 neutralizing antibody improved efferocytosis, vascular integrity and survival rate in sepsis. Collectively, our findings indicated that STAB-1 regulated and maintained vascular integrity through the clearance of infected apoptotic endothelial cells. Moreover, our results suggested that interventions targeting vascular integrity by STAB-1 signalling are promising therapeutic approaches to sepsis.

Combined Rho-kinase inhibition and immunogenic cell death triggers and propagates immunity against cancer.

Activation of T cell immune response is critical for the therapeutic efficacy of cancer immunotherapy. Current immunotherapies have shown remarkable clinical success against several cancers; however, significant responses remain restricted to a minority of patients. Here, we show a therapeutic strategy that combines enhancing the phagocytic activity of antigen-presenting cells with immunogenic cell death to trigger efficient antitumour immunity. Rho-kinase (ROCK) blockade increases cancer cell phagocytosis and induces antitumour immunity through enhancement of T cell priming by dendritic cells (DCs), leading to suppression of tumour growth in syngeneic tumour models. Combining ROCK blockade with immunogenic chemotherapy leads to increased DC maturation and synergistic CD8+ cytotoxic T cell priming and infiltration into tumours. This therapeutic strategy effectively suppresses tumour growth and improves overall survival in a genetic mouse mammary tumour virus/Neu tumour model. Collectively, these results suggest that boosting intrinsic cancer immunity using immunogenic killing and enhanced phagocytosis is a promising therapeutic strategy for cancer immunotherapy.

Nanocage-Therapeutics Prevailing Phagocytosis and Immunogenic Cell Death Awakens Immunity against Cancer.

A growing appreciation of the relationship between the immune system and the tumorigenesis has led to the development of strategies aimed at "re-editing" the immune system to kill tumors. Here, a novel tactic is reported for overcoming the activation-energy threshold of the immunosuppressive tumor microenvironment and mediating the delivery and presentation of tumor neoantigens to the host's immune system. This nature-derived nanocage not only efficiently presents ligands that enhance cancer cell phagocytosis, but also delivers drugs that induce immunogenic cancer cell death. The designed nanocage-therapeutics induce the release of neoantigens and danger signals in dying tumor cells, and leads to enhancement of tumor cell phagocytosis and cross-priming of tumor specific T cells by neoantigen peptide-loaded antigen-presenting cells. Potent inhibition of tumor growth and complete eradication of tumors is observed through systemic tumor-specific T cell responses in tumor draining lymph nodes and the spleen and further, infiltration of CD8+ T cells into the tumor site. Remarkably, after removal of the primary tumor, all mice treated with this nanocage-therapeutics are protected against subsequent challenge with the same tumor cells, suggesting development of lasting, tumor-specific responses. This designed nanocage-therapeutics "awakens" the host's immune system and provokes a durable systemic immune response against cancer.

Homotypic Interaction of Stabilin-2 Plays a Critical Role in Lymph Node Metastasis of Tongue Cancer.

BACKGROUND/AIM: Lymph node (LN) metastasis of solid types of tumors has important clinical significance and it is therefore critical to identify molecular biomarkers that would enable the selection of patients with LN metastases. PATIENTS AND METHODS: We evaluated the expression of stabilin-2 in primary oral tongue tumors and metastatic LNs using immunohistochemical staining. The correlation between risk factors and nodal metastasis was assessed and disease-free survival was analyzed. RESULTS: Stabilin-2 expression remained a significant predictor of LN metastasis and the factor affecting recurrence in tongue cancer. Most importantly, all metastatic tumors of tongue, lung, stomach and colon cancers stained positive for stabilin-2 and stabilin-2 was expressed strongly in the sinusoidal endothelial cell of metastatic LNs. CONCLUSION: Stabilin-2 can play a critical role in the first entrapping step of LN metastasis through homotypic interaction with the lymphatic endothelium and appears to be a tumor biomarker predicting for LN metastasis in patients with solid tumors.

Saturated fatty acid-induced miR-195 impairs insulin signaling and glycogen metabolism in HepG2 cells.

MicroRNAs (miRNAs) play an important role in insulin signaling and insulin secretion, but the role of miRNAs in the association between obesity and hepatic insulin resistance is largely unknown. This study reports that saturated fatty acid (SFA) and high fat diet (HFD) significantly induce miR-195 expression in hepatocytes, and that the insulin receptor (INSR), not insulin receptor substrate-1 (IRS-1), is a direct target of miR-195. Furthermore, the ectopic expression of miR-195 suppresses the expression of INSR, thereby impairing the insulin signaling cascade and glycogen synthesis in HepG2 cells. These findings suggest that the dysregulation of miR-195 by SFA is a detrimental factor for hepatic insulin sensitivity.

Persistent low level of osterix accelerates interleukin-6 production and impairs regeneration after tissue injury.

Osterix (Osx) is an essential transcription factor for osteoblast differentiation and bone formation. Osx knockout show a complete absence of bone formation, whereas Osx conditional knockout in osteoblasts produce an osteopenic phenotype after birth. Here, we questioned whether Osx has a potential role in regulating physiological homeostasis. In Osx heterozygotes expressing low levels of Osx in bones, the expression levels of pro-inflammatory cytokines were significantly elevated, indicating that reduced Osx expression may reflect an inflammatory-prone state. In particular, the expression of interleukin-6, a key mediator of chronic inflammation, was increased in Osx heterozygotes and decreased in Osx overexpressing osteoblasts, and transcriptionally down-regulated by Osx. Although no significant differences were revealed in renal morphology and function between Osx heterozygotes and wild-type under normoxic conditions, recovery of kidneys after ischemic damage was remarkably delayed in Osx heterozygotes, as indicated by elevated blood urea nitrogen and creatinine levels, and by morphological alterations consistent with acute tubular necrosis. Eventually, protracted low Osx expression level caused an inflammatory-prone state in the body, resulting in the enhanced susceptibility to renal injury and the delayed renal repair after ischemia/reperfusion. This study suggests that the maintenance of Osx expression in bone is important in terms of preventing the onset of an inflammatory-prone state.

The induction of miR-96 by mitochondrial dysfunction causes impaired glycogen synthesis through translational repression of IRS-1 in SK-Hep1 cells.

MicroRNA (miRNA) is a class of endogenous small noncoding RNA that negatively regulates gene expression at the post-transcriptional level and plays an important role in the pathogenesis of various diseases. However, the identity and role of miRNAs involved in the development of insulin resistance resulting from mitochondrial dysfunction are largely unknown. In this study, mitochondrial dysfunction by genetic or metabolic inhibition induced an impairment of insulin signaling in SK-Hep1 cells via a reduction in the expression of IRS-1 protein. Significant up-regulation of miR-96, which was presumed to target IRS-1 3'UTR, was found in SK-Hep1 cells with mitochondrial dysfunction. Using reporter gene assay we confirmed that miR-96 authentically targeted IRS-1 3'UTR. Furthermore, the ectopic expression of miR-96 caused a substantial decrease in IRS-1 protein expression, and a consequent impairment in insulin signaling. These findings suggest that the up-regulation of miR-96 by mitochondrial dysfunction contributes to the development of insulin resistance by targeting IRS-1 in SK-Hep1 cells.

Identification of macrophage genes responsive to extracellular acidification.

OBJECTIVE: A low pH microenvironment is a characteristic feature of inflammation loci and affects the functions of immune cells. In this study, we investigated the effect of extracellular acidification on macrophage gene expression. METHODS: RAW264.7 macrophages were incubated in neutral (pH 7.4) or acidic (pH 6.8) medium for 4 h. Global mRNA expression levels were determined using Affymetrix genechips. RESULTS: The mRNA expressions of 353 macrophage genes were significantly modified after incubation in acidic medium; 193 were up-regulated and 160 down-regulated. Differentially regulated genes were grouped into 13 classes based on the functions of the corresponding protein products. Pathway analysis revealed that differentially expressed genes are enriched in pathways related to inflammation and immune responses. Quantitative real-time PCR analysis confirmed that the expressions of CXCL10, CXCL14, IL-18, IL-4RA, ABCA1, CCL4, IL-7R, CXCR4, TLR7, and CCL3 mRNAs were regulated by extracellular acidification. CONCLUSION: The results of this study provide insights into the effects of acidic extracellular environments on macrophage gene expression.

Ets-2 is involved in transcriptional regulation of C1qTNF-related protein 5 in muscle cells.

It was recently reported that C1qTNF-related protein 5 (CTRP5) regulates glucose and lipid metabolism in muscle cells. However, the molecular mechanism of CTRP5 expression has not been fully investigated. In this study we demonstrate the mechanism regulating the transcription of the CTRP5 gene in muscle cells. We found that potential binding sites for transcription factors were conserved in proximal region of CTRP5 promoters in human, mouse and rat. Among these factors, Ets-2 was found to increase the promoter activity of the CTRP5 gene in C2C12 cells. Deletion of -166 to -80 in the CTRP5 promoter region significantly decreased Ets-2-induced CTRP5 promoter activity. Mutagenesis evaluation indicated that two putative Ets-2-binding sites in the CTRP5 promoter are important in CTRP5 activation. Promoter enzyme immunoassay showed that Ets-2 binds directly to two Ets-2-responsive elements at regions -166/-80 of the CTRP5 promoter. Taken together, these results suggest that Ets-2 play a key role in transcriptional regulation of CTRP5 in muscle cells.

Regulation of the transcriptional activation of CTRP3 in chondrocytes by c-Jun.

Although C1qTNF-related protein 3 (CTRP3) is highly expressed in cartilage and is involved in the proliferation and migration of chondrocytes, the molecular mechanism governing the transcriptional regulation of CTRP3 in chondrocytes is largely unknown. Here, we demonstrated the mechanism of transcriptional activation of the CTRP3 gene in chondrogenic cells. We observed that c-Jun stimulated CTRP3 promoter activity in ATDC5 cells. Deletion analysis of the CTRP3 promoter showed that the c-Jun-responsive region is localized at the position between -359 and -176. Mutation of the AP-1 binding site in this region eliminated c-Jun-mediated CTRP3 promoter activity. A promoter enzyme immunoassay and a chromatin immunoprecipitation assay showed that c-Jun binds directly to the AP-1 binding site at the region -184/-177 of the mouse CTRP3 promoter. Thus, these results demonstrated that c-Jun is a cis-acting element for CTRP3 regulation in chondrogenic cells.

MEGF10 functions as a receptor for the uptake of amyloid-ß.

MEGF10 is predominantly expressed in the brain and known to function as a phagocytic receptor. Here, we provide evidence that MEGF10 is involved in the uptake of amyloid-ß peptide (Aß42) in the brain. Overexpression of MEGF10 dramatically increased Aß42 uptake in Hela cells. Knockdown of endogenous MEGF10 expression significantly decreased Aß42 uptake in N2A neuroblastoma cells. MEGF10-mediated Aß uptake is mostly dependent on lipid raft endocytosis pathway. Furthermore, site-directed mutagenesis revealed that the conserved cytoplasmic NPxY and YxxØ motifs are crucial for MEGF10-mediated uptake of Aß42 peptide. Thus, the identification of the MEGF10 as a functional receptor that mediates the uptake of amyloid-ß peptide will help elucidate the molecular mechanisms of amlyoid-ß clearance in Alzheimer's disease.

C1qTNF-related protein-6 increases the expression of interleukin-10 in macrophages.

C1qTNF-Related proteins (CTRPs), a new highly conserved family of adiponectin paralogs, were recently identified as being involved in diverse processes including metabolism, host defense, apoptosis, cell differentiation, and organogenesis. However, the functional role of CTRP6 remains poorly identified. Here we provide evidence that CTRP6 induces the expression of interleukin-10 (IL-10) in macrophages. Conditioned medium from CTRP6-expressing HEK293 cells increased IL-10 expression in Raw264.7 cells. The globular domain of CTRP6 (gCTRP6) also dose-dependently increased both IL-10 mRNA and protein expression levels, with transcript levels increasing within 2 h. Furthermore, the globular domain of CTRP6 rapidly induced phosphorylation of ERK1/2 in Raw264.7 cells. Treatment with U0126, a selective inhibitor, abolished CTRP6-stimulated IL-10 induction. Taken together, there results demonstrate that CTRP6 induces expression of IL-10 via ERK1/2 activation. Considering that IL-10 is a potent anti-inflammatory cytokine that modulates inflammatory signaling pathways, CTRP6 may be a novel target for pharmacological drugs in inflammatory diseases.

The conserved histidine in epidermal growth factor-like domains of stabilin-2 modulates pH-dependent recognition of phosphatidylserine in apoptotic cells.

Clearance of apoptotic cells is involved in the resolution of inflammation, and this mechanism is controlled by the regulation of pro- and anti-inflammatory cytokine production during the ingestion of apoptotic cells. Inflamed areas show extracellular acidity, and low pH stimulates cellular functions of immune cells. However, little is known about the influence of extracellular acidic pH on the function of phagocytic cells. In this study, we showed that stabilin-2-mediated phagocytosis is activated in low pH media (pH 6.8) and examined the molecular mechanisms underlying this pH-dependent enhancement of phagocytic activity. Stabilin-2, which is expressed in human monocyte derived macrophages (HMDM), is a phosphatidylserine (PS) receptor that mediates phagocytosis of apoptotic cells, and releases the anti-inflammatory cytokine, TGF-beta. The PS binding activity of stabilin-2 is enhanced in low pH, and a conserved histidine(1403) in close proximity to the PS binding loop is critical for pH-dependent activity. We propose that protonation of His(1403) may rearrange the PS binding loop to enhance binding affinity in low pH, indicating that acidic pH might act as a danger signal to stimulate stabilin-2-mediated phagocytosis to resolve inflammation. Considering that phosphatidylserine is an important target molecule for apoptotic cells in the acidic microenvironment of inflammation and tumors, our results also have implications for pH sensitive targeting of apoptotic cells.

Human sodium iodide symporter added to multidrug resistance 1 small hairpin RNA in a single gene construct enhances the therapeutic effects of radioiodine in a nude mouse model of multidrug resistant colon cancer.

The objective of this study was to investigate the therapeutic potential of ¹³¹I added to doxorubicin therapy in multidrug resistance (MDR) mouse colon cancer coexpressing the MDR1 small hairpin RNA (shRNA) and human sodium iodide symporter (hNIS) gene in a single gene construct and to visualize the antitumor effects using molecular nuclear imaging. HCT-15 coexpressing shRNA for MDR1 gene (MDR1 shRNA) and hNIS gene with a single construct was established (referred to as MN61 cell). Inhibition of P-gp function by MDR1 shRNA and functional activity of hNIS gene was assessed using a 99(m)Tc sestamibi uptake and ¹²5I uptake, respectively. Cytotoxic effects by a combination of doxorubicin and ¹³¹I were determined in parental (HCT-15) or MN61 cells using an in vitro clonogenic assay. Therapeutic effect of either combination therapy (doxorubicin and ¹³¹I) or single therapy (doxorubicin or ¹³¹I alone) was evaluated by tumor volume measurement. 99(m)Tc-sestamibi, ¹²³I, and 99(m)Tc-pertechnetate images of mice were acquired to evaluate functional assessment in vivo. Cellular uptake of 99(m)Tc-sestamibi and ¹²5I was approximately 2-fold and 100-fold higher in MN61 cells than in parental cells, respectively. Combination of ¹³¹I and doxorubicin resulted in higher cytotoxcity in MN61 cells as compared with parental cells. Scintigraphic imaging showed higher uptake of 99(m)Tc-sestamibi and ¹²³I in MN61 tumor as compared with parental tumor. In mice treated with doxorubicin, there was a slight delay in tumor growth in the MN61 tumor but not in the parental tumor. Cancer treatment with ¹³¹I or doxorubicin induced a rapid reduction of tumor volume in the MN61 tumor but not in the parental tumor. Combination therapy further generated a rapid reduction of tumor volume as compared with ¹³¹I therapy alone (p < 0.05). A combination hNIS mediated radioiodine gene therapy added to MDR1 shRNA treatment improved the effects of cancer treatment in a MDR cancer model and could enable visualization of the antitumor effects with nuclear imaging.