Valproic acid potentiates the anticancer activity of capecitabine in vitro and in vivo in breast cancer models via induction of thymidine phosphorylase expression.
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ABSTRACT: The prognosis of patients with metastatic breast cancer remains poor, and thus novel therapeutic approaches are needed. Capecitabine, which is commonly used for metastatic breast cancer in different settings, is an inactive prodrug that takes advantage of elevated levels of thymidine phosphorylase (TP), a key enzyme that is required for its conversion to 5-fluororacil, in tumors. We demonstrated that histone deacetylase inhibitors (HDACi), including low anticonvulsant dosage of VPA, induced the dose- and time-dependent up-regulation of TP transcript and protein expression in breast cancer cells, but not in the non-tumorigenic breast MCF-10A cell line. Through the use of siRNA or isoform-specific HDACi, we demonstrated that HDAC3 is the main isoform whose inhibition is involved in the modulation of TP. The combined treatment with capecitabine and HDACi, including valproic acid (VPA), resulted in synergistic/additive antiproliferative and pro-apoptotic effects in breast cancer cells but not in TP-knockout cells, both in vitro and in vivo, highlighting the crucial role of TP in the synergism observed. Overall, this study suggests that the combination of HDACi (e.g., VPA) and capecitabine is an innovative antitumor strategy that warrants further clinical evaluation for the treatment of metastatic breast cancer.
Project description:Thymidine phosphorylase (TP) catalyzes the conversion of thymidine to thymine and 2-deoxyribose-1-phosphate. The latter plays an important role in induction of angiogenesis. As such, many human malignancies exhibit TP overexpression that correlates with increased microvessel density, formation of aggressive tumors, and dismal prognosis. Because TP is frequently overexpressed in cancer, pro-drugs were developed that utilize TP activity for their bioactivation to cytotoxic drugs. In this respect, TP is indispensable for the pharmacologic activity of the chemotherapeutic drug capecitabine, as it converts its intermediary metabolite 5'-deoxyfluorouridine to 5-fluorouracil. Thus, loss of TP function confers resistance to the prodrug capecitabine, currently used for the treatment of metastatic colorectal cancer and breast cancer. However, drug resistance phenomena may frequently emerge that compromise the pharmacologic activity of capecitabine. Deciphering the molecular mechanisms underlying resistance to TP-activated prodrugs is an important goal toward the overcoming of such drug resistance phenomena. Here, we discovered that lack of TP protein in drug-resistant tumor cells is due to unsplicing of its pre-mRNA. Advanced bioinformatics identified the family of heterogeneous nuclear ribonucleoproteins (hnRNP) H/F as candidate splicing factors potentially responsible for impaired TP splicing. Indeed, whereas parental cells lacked nuclear localization of hnRNPs H1/H2 and F, drug-resistant cells harbored marked levels of these splicing factors. Nuclear RNA immunoprecipitation experiments established a strong binding of hnRNP H1/H2 to TP pre-mRNA, hence implicating them in TP splicing. Moreover, introduction of hnRNP H2 into drug-sensitive parental cells recapitulated aberrant TP splicing and 5'-deoxyfluorouridine resistance. Thus, this is the first study identifying altered function of hnRNP H1/H2 in tumor cells as a novel determinant of aberrant TP splicing thereby resulting in acquired chemoresistance to TP-activated fluoropyrimidine anticancer drugs.
Project description:Human thymidine phosphorylase (hTP) is responsible for thymidine (dT) homeostasis, and its action promotes angiogenesis. In the absence of phosphate, hTP catalyzes a slow hydrolytic depyrimidination of dT yielding thymine and 2-deoxyribose (dRib). Its transition state was characterized using multiple kinetic isotope effect (KIE) measurements. Isotopically enriched thymidines were synthesized enzymatically from glucose or (deoxy)ribose, and intrinsic KIEs were used to interpret the transition state structure. KIEs from [1'-(14)C]-, [1-(15)N]-, [1'-(3)H]-, [2'R-(3)H]-, [2'S-(3)H]-, [4'-(3)H]-, and [5'-(3)H]dTs provided values of 1.033 ± 0.002, 1.004 ± 0.002, 1.325 ± 0.003, 1.101 ± 0.004, 1.087 ± 0.005, 1.040 ± 0.003, and 1.033 ± 0.003, respectively. Transition state analysis revealed a stepwise mechanism with a 2-deoxyribocation formed early and a higher energetic barrier for nucleophilic attack of a water molecule on the high energy intermediate. An equilibrium exists between the deoxyribocation and reactants prior to the irreversible nucleophilic attack by water. The results establish activation of the thymine leaving group without requirement for phosphate. A transition state constrained to match the intrinsic KIEs was found using density functional theory. An active site histidine (His116) is implicated as the catalytic base for activation of the water nucleophile at the rate-limiting transition state. The distance between the water nucleophile and the anomeric carbon (r(C-O)) is predicted to be 2.3 A at the transition state. The transition state model predicts that deoxyribose adopts a mild 3'-endo conformation during nucleophilic capture. These results differ from the concerted bimolecular mechanism reported for the arsenolytic reaction (Birck, M. R.; Schramm, V. L. J. Am. Chem. Soc. 2004, 126, 2447-2453).
Project description:ObjectiveThymidine Phosphorylase (TYMP) gene was of potential significance in the process of colorectal cancer (CRC) development and played an important role in capecitabine metabolism. This study was to identify the association between TYMP polymorphism and prognosis of postoperative patients with CRC who received capecitabine-based adjuvant chemotherapy.MethodsA total of 218 patients with CRC who were treated with surgical resection and capecitabine-based adjuvant chemotherapy were included in this study retrospectively. Peripheral blood and peripheral blood mononuclear cell (PBMC) specimen of the patients were collected for the genotyping of TYMP polymorphism and TYMP mRNA expression, respectively. Univariate analysis of genotypes and prognosis was carried out by Kaplan-Meier survival analysis, Cox regression analysis was adopted in multivariate analysis. The mRNA expression of TYMP according to genotype status was analyzed using non-parameter test.ResultsPrevalence of rs11479 in TYMP among the 218 patients exhibited that minor allele frequency of rs11479 was 0.20 (GG 141 cases, GA 68 cases and AA 9 cases), which was in accordance with Hardy-Weinberg equilibrium (P=0.825). Association analysis suggested that the median disease-free survival (DFS) of patients with GG genotype and GA/AA genotype was 3.1 and 6.1 years, respectively (P=0.004). Furthermore, the median overall survival of patients with GG genotype and GA/AA genotype was 5.0 and 7.0 years, respectively (P=0.033). Multivariate Cox regression analysis exhibited that rs11479 polymorphism was an independent factor for DFS (HR = 1.64, P=0.009). Additionally, of the 65 PBMC specimens, mRNA expression results indicated that patients with GA/AA genotypes conferred significantly higher mRNA expression of TYMP than that of patients with GG genotype (P<0.001).ConclusionPolymorphism rs11479 in TYMP gene might predict the prognosis of patients with CRC who received capecitabine-based adjuvant chemotherapy through mediation of the mRNA expression of TYMP. The conclusion of this study should be validated in prospective clinical trials subsequently.
Project description:KP1339 is a promising ruthenium-based anticancer compound in early clinical development. This study aimed to test the effects of KP1339 on the in vitro and in vivo activity of the multi-kinase inhibitor sorafenib, the current standard first-line therapy for advanced hepatoma. Anticancer activity of the parental compounds as compared to the drug combination was tested against a panel of cancer cell lines with a focus on hepatoma. Combination of KP1339 with sorafenib induced in the majority of all cases distinctly synergistic effects, comprising both sorafenib-resistant as well as sorafenib-responsive cell models. Several mechanisms were found to underlie these multifaceted synergistic activities. Firstly, co-exposure induced significantly enhanced accumulation levels of both drugs resulting in enhanced apoptosis induction. Secondly, sorafenib blocked KP1339-mediated activation of P38 signalling representing a protective response against the ruthenium drug. In addition, sorafenib treatment also abrogated KP1339-induced G2/M arrest but resulted in check point-independent DNA-synthesis block and a complete loss of the mitotic cell populations. The activity of the KP1339/sorafenib combination was evaluated in the Hep3B hepatoma xenograft. KP1339 monotherapy led to a 2.4-fold increase in life span and, thus, was superior to sorafenib, which induced a 1.9-fold prolonged survival. The combined therapy further enhanced the mean survival by 3.9-fold. Synergistic activity was also observed in the VM-1 melanoma xenograft harbouring an activating braf mutation. Together, our data indicate that the combination of KP1339 with sorafenib displays promising activity in vitro and in vivo especially against human hepatoma models.
Project description:Cervical cancer is globally the fourth most common cancer in women. Metformin is a widely used drug for the treatment of type II diabetes and has been shown to possess important anticancer properties in cervical cancer. Everolimus is an mTOR inhibitor and is widely used to treat NETs, RCC, TSC, and breast cancers. The present study investigated the anticancer effects of metformin and everolimus in cervical cancer, when used alone or in combination. CaSki and C33A human cervical cancer cells were treated with different concentrations of everolimus alone or in combination with metformin. Cell viability was assessed using a CCK-8 assay. Cell apoptosis, cell-cycle, and mtROS analyses were conducted using flow cytometry. Target protein levels were analyzed by Western blotting. Related mechanisms were confirmed using appropriate inhibitors (z-VAD-fmk and BIRB796). The in vitro results were further confirmed in a xenograft tumor study. Both metformin and everolimus, when used alone, were moderately effective in inhibiting cell proliferation and inducing cell apoptosis of CaSki and C33A cells. When used in combination, these two drugs synergistically inhibited the growth of human cervical cancer cells and xenografts in nude mice, promoted sub-G1- and G0/G1-phase cell-cycle arrest, and enhanced mtROS production. The protein expressions of PI3K (p110α) and p-AKT were significantly downregulated, while P27, P21, p-p38, p-ERK, and p-JNK were upregulated following combined treatment. These results revealed that metformin potentiates the anticancer effect of everolimus on cervical cancer, and combination treatment with metformin and everolimus provides a novel therapeutic strategy for patients with cervical cancer.
Project description:Paclitaxel (PTX) has been the first-line treatment for lung cancer; however, its clinical use is limited due to multidrug resistance (MDR) and adverse effects. Thus, there is an urgent need to explore agents that can enhance the anticancer efficacy of PTX by reducing drug resistance and adverse reactions. Jiegeng decoction (JG) was used as the meridian guide drug and adjuvant drug in treatment of lung cancer. However, the mechanism of adjuvant effect was unclear. The aim of this study was to determine whether JG could potentiate the anticancer effect of PTX. Tissue distribution of PTX was detected using HPLC-MS/MS. The anti-lung cancer effect of the combination of PTX and JG in Lewis lung cancer C57BL/6J mice was evaluated based on the body weight and tumor-inhibition rate. PTX concentration in tumors was determined using HPLC-MS and in vivo imaging. Biochemical indices were detected using biochemical analyzer and ELISA. The anticancer mechanism of the PTX-JG combination in A549/PTX cells was elucidated based on cell proliferation, annexin V-FITC apoptosis assay, and western blotting. Tissue distribution analysis showed that the distribution of PTX increased in the lungs, liver, and heart upon administering the combination of PTX and JG. JG remarkably enhanced the anticancer effect of PTX by increasing the red blood cell and platelet counts; increasing hemoglobin, interleukin (IL)-2, and tumor necrosis factor-α levels; increasing CD4+T cells and the CD4+/CD8+ ratio; and decreasing IL-10 levels. JG administration led to the increased distribution of PTX at the tumor lesion sites and also potentiated the anticancer effect of PTX by inhibiting tumor cell proliferation and promoting apoptosis. Moreover, JG reversed PTX resistance by inhibiting the expression of lung resistance-related proteins, multiresistance protein 1, P-glycoprotein, and breast cancer-resistant protein. Furthermore, the combination of JG and PTX decreased alanine aminotransferase and aspartate aminotransferase levels and did not affect creatine kinase-MB levels. Therefore, our discovery suggests that JG increased the anticancer effect of PTX by downregulating the MDR-related protein and demonstrated a synergistic enhancement of immunity. Thus, the combination of PTX with JG shows potential in the management of lung cancer owing to its synergistic and detoxifying effects.
Project description:Human thymidine phosphorylase (hTP) is responsible for thymidine (dT) homeostasis, promotes angiogenesis, and is involved in metabolic inactivation of antiproliferative agents that inhibit thymidylate synthase. Understanding its transition state structure is on the path to design transition state analogues. Arsenolysis of dT by hTP permits kinetic isotope effect (KIE) analysis of the reaction by forming thymine and the chemically unstable 2-deoxyribose 1-arsenate. The transition state for the arsenolytic reaction was characterized using multiple KIEs and computational analysis. Transition state analysis revealed a concerted bimolecular (A(N)D(N)) mechanism. A transition state constrained to match the intrinsic KIE values was found using density functional theory (B3LYP/6-31G*). An active site histidine is implicated as the catalytic base responsible for activation of the arsenate nucleophile and stabilization of the thymine leaving group during the isotopically sensitive step. At the transition state, the deoxyribose ring exhibits significant oxocarbenium ion character with bond breaking (r(C-N) = 2.45 Å) nearly complete and minimal bond making to the attacking nucleophile (r(C-O) = 2.95 Å). The transition state model predicts a deoxyribose conformation with a 2'-endo ring geometry. Transition state structure for the slow hydrolytic reaction of hTP involves a stepwise mechanism [Schwartz, P. A., Vetticatt, M. J., and Schramm, V. L. (2010) J. Am. Chem. Soc. 132, 13425-13433], in contrast to the concerted mechanism described here for arsenolysis.
Project description:Thymidine phosphorylase (TP) regulates intracellular and plasma thymidine levels. TP deficiency is hypothesized to (i) increase levels of thymidine in plasma, (ii) lead to mitochondrial DNA alterations, and (iii) cause mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). In order to elucidate the physiological roles of TP, we generated mice deficient in the TP gene. Although TP activity in the liver was inhibited in these mice, it was fully maintained in the small intestine. Murine uridine phosphorylase (UP), unlike human UP, cleaves thymidine, as well as uridine. We therefore generated TP-UP double-knockout (TP(-/-) UP(-/-)) mice. TP activities were inhibited in TP(-/-) UP(-/-) mice, and the level of thymidine in the plasma of TP(-/-) UP(-/-) mice was higher than for TP(-/-) mice. Unexpectedly, we could not observe alterations of mitochondrial DNA or pathological changes in the muscles of the TP(-/-) UP(-/-) mice, even when these mice were fed thymidine for 7 months. However, we did find hyperintense lesions on magnetic resonance T(2) maps in the brain and axonal edema by electron microscopic study of the brain in TP(-/-) UP(-/-) mice. These findings suggested that the inhibition of TP activity caused the elevation of pyrimidine levels in plasma and consequent axonal swelling in the brains of mice. Since lesions in the brain do not appear to be due to mitochondrial alterations and pathological changes in the muscle were not found, this model will provide further insights into the causes of MNGIE.
Project description:Currently approved immune checkpoint inhibitor therapies targeting the PD-1 and CTLA-4 receptor pathways are powerful treatment options for certain cancers; however, most patients across cancer types still fail to respond. Consequently, there is interest in discovering and blocking alternative pathways that mediate immune suppression. One such mechanism is an upregulation of sialoglycans in malignancy, which has been recently shown to inhibit immune cell activation through multiple mechanisms and therefore represents a targetable glycoimmune checkpoint. Since these glycans are not canonically druggable, we designed an αHER2 antibody-sialidase conjugate that potently and selectively strips diverse sialoglycans from breast cancer cells. In syngeneic breast cancer models, desialylation enhanced immune cell infiltration and activation and prolonged the survival of mice, an effect that was dependent on expression of the Siglec-E checkpoint receptor found on tumor-infiltrating myeloid cells. Thus, antibody-sialidase conjugates represent a promising modality for glycoimmune checkpoint therapy.
Project description:RationalePlatelets contain abundant thymidine phosphorylase (TYMP), which is highly expressed in diseases with high risk of thrombosis, such as atherosclerosis and type II diabetes mellitus.ObjectiveTo test the hypothesis that TYMP participates in platelet signaling and promotes thrombosis.Methods and resultsBy using a ferric chloride (FeCl3)-induced carotid artery injury thrombosis model, we found time to blood flow cessation was significantly prolonged in Tymp(-/-) and Tymp(+/-) mice compared with wild-type mice. Bone marrow transplantation and platelet transfusion studies demonstrated that platelet TYMP was responsible for the antithrombotic phenomenon in the TYMP-deficient mice. Collagen-, collagen-related peptide-, adenosine diphosphate-, or thrombin-induced platelet aggregation were significantly attenuated in Tymp(+/-) and Tymp(-/-) platelets, and in wild type or human platelets pretreated with TYMP inhibitor KIN59. Tymp deficiency also significantly decreased agonist-induced P-selectin expression. TYMP contains an N-terminal SH3 domain-binding proline-rich motif and forms a complex with the tyrosine kinases Lyn, Fyn, and Yes in platelets. TYMP-associated Lyn was inactive in resting platelets, and TYMP trapped and diminished active Lyn after collagen stimulation. Tymp/Lyn double haploinsufficiency diminished the antithrombotic phenotype of Tymp(+/-) mice. TYMP deletion or inhibition of TYMP with KIN59 dramatically increased platelet-endothelial cell adhesion molecule 1 tyrosine phosphorylation and diminished collagen-related peptide- or collagen-induced AKT phosphorylation. In vivo administration of KIN59 significantly inhibited FeCl3-induced carotid artery thrombosis without affecting hemostasis.ConclusionsTYMP participates in multiple platelet signaling pathways and regulates platelet activation and thrombosis. Targeting TYMP might be a novel antiplatelet and antithrombosis therapy.