RAC2-P38 MAPK-dependent NADPH oxidase activity is associated with the resistance of quiescent cells to ionizing radiation

Our recent study showed that quiescent G0 cells are more resistant to ionizing radiation than G1 cells; however, the underlying mechanism for this increased radioresistance is unknown. Based on the relatively lower DNA damage induced in G0 cells, we hypothesize that these cells are exposed to less oxidative stress during exposure. As a catalytic subunit of NADPH oxidase, Ras-related C3 botulinum toxin substrate 2 (RAC2) may be involved in the cellular response to ionizing radiation. Here, we show that RAC2 was expressed at low levels in G0 cells but increased substantially in G1 cells. Relative to G1 cells, the total antioxidant capacity in G0 phase cells increased upon exposure to X-ray radiation, whereas the intracellular concentration of ROS and malondialdehyde increased only slightly. The induction of DNA single- and double-stranded breaks in G1 cells by X-ray radiation was inhibited by knockdown of RAC2. P38 MAPK interaction with RAC2 resulted in a decrease of functional RAC2. Increased phosphorylation of P38 MAPK in G0 cells also increased cellular radioresistance; however, excessive production of ROS caused P38 MAPK dephosphorylation. P38 MAPK, phosphorylated P38 MAPK, and RAC2 regulated in mutual feedback and negative feedback regulatory pathways, resulting in the radioresistance of G0 cells.     

Chloride intracellular channel 1 identified using proteomic analysis plays an important role in the radiosensitivity of HEp‐2 cells via reactive oxygen species production

The nature of the molecules underlying the radioresistance phenotype of laryngeal cancer cells remains to be established. We initially generated radioresistant laryngeal cancer cell lines from human HEp‐2 cells with fractionated radiation. These RR‐HEp‐2 cells and isolated clones displayed more radioresistant and anti‐apoptotic phenotypes than parental HEp‐2 cells after radiation. Characteristics of RR‐Hep‐2 cell lines were confirmed by upregulation of radioresistance‐related genes, such as epidermal growth factor receptor, Hsp90, and Bcl‐xl. Subsequently, we examined proteome changes between HEp‐2 and RR‐HEp‐2 cells and identified 16 proteins showing significantly altered expression levels. Interestingly, protein expression of chloride intracellular channel 1 (CLIC1) was markedly suppressed in RR‐HEp‐2 cells, compared with non‐irradiated control cells. Suppression of CLIC1 with an indanyloxyacetic acid‐94 or small interfering RNA led to radioresistance in HEp‐2 cells by suppressing the radiation‐induced cellular ROS level. However, ectopic overexpression of CLIC1 induced radiosensitivity in RR‐HEp‐2 cells via induction of ROS level after radiation, suggesting that the protein acts as a positive regulator of ROS production. Our results collectively indicate that suppression of CLIC1 contributes to acquisition of the radioresistance phenotype of laryngeal cancer cells via inhibition of ROS production, implying that this protein is an important candidate molecule for radiotherapy in radioresistant laryngeal cancer cells.     

Increased MCL-1 synthesis promotes irradiation-induced nasopharyngeal carcinoma radioresistance via regulation of the ROS/AKT loop

Worldwide, nasopharyngeal carcinoma (NPC) is a rare head and neck cancer; however, it is a common malignancy in southern China. Radiotherapy is the most important treatment strategy for NPC. However, although radiotherapy is a strong tool to kill cancer cells, paradoxically it also promotes aggressive phenotypes. Therefore, we mimicked the treatment process in NPC cells in vitro. Upon exposure to radiation, a subpopulation of NPC cells gradually developed resistance to radiation and displayed cancer stem-cell characteristics. Radiation-induced stemness largely depends on the accumulation of the antiapoptotic myeloid cell leukemia 1 (MCL-1) protein. Upregulated MCL-1 levels were caused by increased stability and more importantly, enhanced protein synthesis. We showed that repeated ionizing radiation resulted in persistently enhanced reactive oxygen species (ROS) production at a higher basal level, further promoting protein kinase B (AKT) signaling activation. Intracellular ROS and AKT activation form a positive feedback loop in the process of MCL-1 protein synthesis, which in turn induces stemness and radioresistance. AKT/MCL-1 axis inhibition attenuated radiation-induced resistance, providing a potential target to reverse radiation therapy-induced radioresistance.Subject terms: Experimental models of disease, Radiotherapy, Head and neck cancer     

AKR1C1 promotes non-small cell lung cancer proliferation via crosstalk between HIF-1α and metabolic reprogramming

Non-small cell lung cancer (NSCLC) ranks first among cancer death worldwide. Despite efficacy and safety priority, targeted therapy only benefits ∼30% patients, leading to the unchanged survival rates for whole NSCLC patients. Metabolic reprogramming occurs to offer energy and intermediates for fuelling cancer cells proliferation. Thus, mechanistic insights into metabolic reprogramming may shed light upon NSCLC proliferation and find new proper targets for NSCLC treatment. Herein, we used loss- and gain-of-function experiments to uncover that highly expressed aldo-keto reductase family1 member C1 (AKR1C1) accelerated NSCLC cells proliferation via metabolic reprogramming. Further molecular profiling analyses demonstrated that AKR1C1 augmented the expression of hypoxia-inducible factor 1-alpha (HIF-1α), which could drive tumour metabolic reprogramming. What's more, AKR1C1 significantly correlated with HIF-1α signaling, which predicted poor prognosis for NSCLC patients. Collectively, our data display that AKR1C1 reprograms tumour metabolism to promote NSCLC cells proliferation by activating HIF-1α. These newly acquired data not only establish the specific role for AKR1C1 in metabolic reprogramming, but also hint to the possibility that AKR1C1 may be a new therapeutic target for NSCLC treatment.     

Involvement of an aldo-keto reductase (AKR1C3) in redox cycling of 9,10-phenanthrenequinone leading to apoptosis in human endothelial cells

9,10-Phenanthrenequinone (9,10-PQ), a major quinone found in diesel exhaust particles, is considered to generate reactive oxygen species (ROS) through its redox cycling. Here, we show that 9,10-PQ evokes apoptosis in human aortic endothelial cells (HAECs) and its apoptotic signaling includes ROS generation and caspase activation. The 9,10-PQ-induced cytotoxicity was inhibited by ROS scavengers, indicating that intracellular ROS generation is responsible for the 9,10-PQ-induced apoptosis. Comparison of mRNA expression levels and kinetic constants in the 9,10-PQ reduction among 10 human reductases suggests that aldo-keto reductase 1C3 (AKR1C3) is a 9,10-PQ reductase in HAECs. In in vitro 9,10-PQ reduction by AKR1C3, the reduced product 9,10-dihydroxyphenanthrene and superoxide anions were formed, suggesting the enzymatic two-electron reduction of 9,10-PQ that thereby causes oxidative stress through its redox cycling. In addition, the participation of AKR1C3 in 9,10-PQ-redox cycling was confirmed by the data that AKR1C3 overexpression in endothelial cells augmented the ROS generation and cytotoxicity by 9,10-PQ, and the ROS scavengers inhibited the toxic effects. Pretreatment of the overexpressing cells with AKR1C3 inhibitors, flufenamic acid and indomethacin, suppressed the 9,10-PQ-induced GSH depletion. These results suggest that AKR1C3 is a key enzyme in the initial step of 9,10-PQ-induced cytotoxicity in HAECs.     

WISP-1 Contributes to Fractionated Irradiation-Induced Radioresistance in Esophageal Carcinoma Cell Lines and Mice

Cancer cells that survive fractionated irradiation can be radioresistant and cause tumor recurrence. However, the molecular mechanisms underlying the development of radioresistance in cancer cells remain elusive. The aim of this study was to investigate the role of WISP-1 in the development of radioresistance in esophageal carcinoma during fractionated irradiation. Radioresistant esophageal cancer cells were generated from normal esophageal cancer cells via fractionated irradiation, and expression levels of related proteins were determined by Western blot. Radiosensitivity of cells was established by clonogenic cell survival assays, and cell cycle distribution was evaluated by flow cytometry. Protein distributions were determined by immunofluorescence, and cell toxicity was evaluated by cell counting kit-8 assays. In vivo validations were performed in a xenograft transplantation mouse model. Our data indicate that WISP-1 plays an important role in the development of radioresistance in esophageal cancer cells during fractionated irradiation. The overexression of WISP-1 in esophageal cancer cells was associated with radioresistance. Depletion of extracellular WISP-1 by antibody neutralizing reversed radioresistance and directly induced mitotic catastrophe resulting in cell death. WISP-1 may be a candidate therapeutic target in the treatment of recurrent esophageal carcinoma after radiotherapy.     

Resveratrol and piperine enhance radiosensitivity of tumor cells

The use of ionizing radiation (IR) is essential for treating many human cancers. However, radioresistance markedly impairs the efficacy of tumor radiotherapy. IR enhances the production of reactive oxygen species (ROS) in a variety of cells which are determinant components in the induction of apoptosis. Much interest has developed to augment the effect of radiation in tumors by combining it with radiosensitizers to improve the therapeutic ratio. In the current study, the radiosensitizing effects of resveratrol and piperine on cancer cells were evaluated. Cancer cell lines treated with these natural products exhibited significantly augmented IR-induced apoptosis and loss of mitochondrial membrane potential, presumably through enhanced ROS generation. Applying natural products as sensitizers for IR-induced apoptotic cell death offers a promising therapeutic approach to treat cancer.     

9,10-Phenanthrenequinone promotes secretion of pulmonary aldo-keto reductases with surfactant

9,10-Phenanthrenequinone (9,10-PQ), a major quinone in diesel exhaust particles, induces apoptosis via the generation of reactive oxygen species (ROS) because of 9,10-PQ redox cycling. We have found that intratracheal infusion of 9,10-PQ facilitates the secretion of surfactant into rat alveolus. In the cultured rat lung, treatment with 9,10-PQ results in an increase in a lower-density surfactant by ROS generation through redox cycling of the quinone. The surfactant contains aldo-keto reductase (AKR) 1C15, which reduces 9,10-PQ and the enzyme level in the surfactant increases on treatment with 9,10-PQ suggesting an involvement of AKR1C15 in the redox cycling of the quinone. In six human cell types (A549, MKN45, Caco2, Hela, Molt4 and U937) only type II epithelial A549 cells secrete three human AKR1C subfamily members (AKR1C1, AKR1C2 and AKR1C3) with the surfactant into the medium; this secretion is highly increased by 9,10-PQ treatment. Using in vitro enzyme inhibition analysis, we have identified AKR1C3 as the most abundantly secreted AKR1C member. The AKR1C enzymes in the medium efficiently reduce 9,10-PQ and initiate its redox cycling accompanied by ROS production. The exposure of A549 cells to 9,10-PQ provokes viability loss, which is significantly protected by the addition of the AKR1C3 inhibitor and antioxidant enzyme and by the removal of the surfactants from the culture medium. Thus, the AKR1C enzymes secreted in pulmonary surfactants probably participate in the toxic mechanism triggered by 9,10-PQ.     

Retinaldehyde is a substrate for human aldo-keto reductases of the 1C subfamily

Human AKR (aldo-keto reductase) 1C proteins (AKR1C1-AKR1C4) exhibit relevant activity with steroids, regulating hormone signalling at the pre-receptor level. In the present study, investigate the activity of the four human AKR1C enzymes with retinol and retinaldehyde. All of the enzymes except AKR1C2 showed retinaldehyde reductase activity with low Km values (~1 μM). The kcat values were also low (0.18-0.6 min-1), except for AKR1C3 reduction of 9-cis-retinaldehyde whose kcat was remarkably higher (13 min-1). Structural modelling of the AKR1C complexes with 9-cis-retinaldehyde indicated a distinct conformation of Trp227, caused by changes in residue 226 that may contribute to the activity differences observed. This was partially supported by the kinetics of the AKR1C3 R226P mutant. Retinol/retinaldehyde conversion, combined with the use of the inhibitor flufenamic acid, indicated a relevant role for endogenous AKR1Cs in retinaldehyde reduction in MCF-7 breast cancer cells. Overexpression of AKR1C proteins depleted RA (retinoic acid) transactivation in HeLa cells treated with retinol. Thus AKR1Cs may decrease RA levels in vivo. Finally, by using lithocholic acid as an AKR1C3 inhibitor and UVI2024 as an RA receptor antagonist, we provide evidence that the pro-proliferative action of AKR1C3 in HL-60 cells involves the RA signalling pathway and that this is in part due to the retinaldehyde reductase activity of AKR1C3.     

AKR1B10 confers resistance to radiotherapy via FFA/TLR4/NF-κB axis in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is one kind of human head and neck cancers with high incidence in Southern China, Southeast Asia and North Africa. In spite of great innovations in radiation and chemotherapy treatments, the 5-year survival rate is not satisfactory. One of the main reasons is resistance to radiotherapy which leads to therapy failure and recurrence of NPC. The mechanism underlying remains to be fully elucidated. Aldo-keto reductase B10 (AKR1B10) plays a role in the formation and development of carcinomas. However, its role in resistance to radiotherapy of NPC is not clear. In this research, the relationships between AKR1B10 expression and the treatment effect of NPC patients, NPC cell survival, cell apoptosis, and DNA damage repair, as well as the effect and mechanism of AKR1B10 expression on NPC radioresistance were explored. A total of 58 paraffin tissues of NPC patients received radiotherapy were collected including 30 patients with radiosensitivity and 28 patients with radioresistance. The relationships between AKR1B10 expression and the treatment effect as well as clinical characteristics were analyzed by immuno-histochemical experiments, and the roles of AKR1B10 in cell survival, apoptosis and DNA damage repair were detected using the AKR1B10 overexpressed cell models. Furthermore the mechanism of AKR1B10 in NPC radioresistance was explored. Finally, the radioresistance effect of AKR1B10 expression was evaluated by the tumor xenograft model of nude mice and the method of radiotherapy. The results showed AKR1B10 expression level was correlated with radiotherapy resistance, and AKR1B10 overexpression promoted proliferation of NPC cells, reduced apoptosis and decreased cellular DNA damage after radiotherapy. The probable molecular mechanism is that AKR1B10 expression activated FFA/TLR4/NF-κB axis in NPC cells. This was validated by using the TLR4 inhibitor TAK242 to treat NPC cells with AKR1B10 expression, which reduced the phosphorylation of NF-κB. This study suggests that AKR1B10 can induce radiotherapy resistance and promote cell survival via FFA/TLR4/NF-κB axis in NPC, which may provide a novel target to fight against radiotherapy resistance of NPC.     

Aldo-keto reductase 1C15 as a quinone reductase in rat endothelial cell: its involvement in redox cycling of 9,10-phenanthrenequinone

9,10-Phenanthrenequinone (9,10-PQ), a redox-active quinone in diesel exhausts, triggers cellular apoptosis via reactive oxygen species (ROS) generation in its redox cycling. This study found that induction of CCAAT/enhancer-binding protein-homologous protein (CHOP), a pro-apoptotic factor derived from endoplasmic reticulum stress, participates in the mechanism of rat endothelial cell damage. The 9,10-PQ-mediated CHOP induction was strengthened by a proteasome inhibitor (MG132) and the MG132-induced cell sensitization to the 9,10-PQ toxicity was abolished by a ROS inhibitor, suggesting that ROS generation and consequent proteasomal dysfunction are responsible for the CHOP up-regulation caused by 9,10-PQ. Aldo-keto reductase (AKR) 1C15 expressed in rat endothelial cells reduced 9,10-PQ into 9,10-dihydroxyphenanthrene concomitantly with superoxide anion formation, implying its participation in evoking the 9,10-PQ-redox cycling. The 9,10-PQ-induced damage was augmented by AKR1C15 over-expression. 9,10-PQ also provoked the AKR1C15 up-regulation, which sensitized against the quinone toxicity. These results suggest the presence of a negative feedback loop exacerbating the quinone toxicity in rat endothelial cells.     

Aldo-keto reductase 1C15 as a quinone reductase in rat endothelial cell: Its involvement in redox cycling of 9,10-phenanthrenequinone

Abstract

9,10-Phenanthrenequinone (9,10-PQ), a redox-active quinone in diesel exhausts, triggers cellular apoptosis via reactive oxygen species (ROS) generation in its redox cycling. This study found that induction of CCAAT/enhancer-binding protein-homologous protein (CHOP), a pro-apoptotic factor derived from endoplasmic reticulum stress, participates in the mechanism of rat endothelial cell damage. The 9,10-PQ-mediated CHOP induction was strengthened by a proteasome inhibitor (MG132) and the MG132-induced cell sensitization to the 9,10-PQ toxicity was abolished by a ROS inhibitor, suggesting that ROS generation and consequent proteasomal dysfunction are responsible for the CHOP up-regulation caused by 9,10-PQ. Aldo-keto reductase (AKR) 1C15 expressed in rat endothelial cells reduced 9,10-PQ into 9,10-dihydroxyphenanthrene concomitantly with superoxide anion formation, implying its participation in evoking the 9,10-PQ-redox cycling. The 9,10-PQ-induced damage was augmented by AKR1C15 over-expression. 9,10-PQ also provoked the AKR1C15 up-regulation, which sensitized against the quinone toxicity. These results suggest the presence of a negative feedback loop exacerbating the quinone toxicity in rat endothelial cells.     

Simvastatin inhibits the development of radioresistant esophageal cancer cells by increasing the radiosensitivity and reversing EMT process via the PTEN-PI3K/AKT pathway

Acquired radioresistance compromises the efficacy of radiotherapy for carcinomas including esophageal cancer (EC), thus resulting in recurrence and poor survival. Recent research corroborated radiosensitive function of simvastatin in stem-like breast cancer cells. However, its role in EC radioresistance remains poorly elucidated. Here, we developed a radioresistant EC cell line Ec9706-R with higher resistance to irradiation relative to control Ec9706 cells. Intriguingly, Ec9706-R cells exhibited epithelial-mesenchymal transition (EMT) characteristics with high invasion and migration ability. Simvastatin sensitized radioresistance of Ec9706-R cells and suppressed cell proliferation, but aggravated radiation-induced apoptosis and caspase-3 activity. Furthermore, simvastatin reversed EMT and inhibited cell invasion and migration of Ec9706-R cells. Mechanism assay confirmed the activation of PI3K/AKT pathway after radiation, which was inhibited by simvastatin. After restoring this pathway by its activator, IGF-1, simvastatin-mediated radiosensitivity and EMT reversion were abrogated. Further assay substantiated the PTEN suppression after irradiation, which was elevated following simvastatin pre-treatment. Moreover, PTEN cessation attenuated the inhibitory effect of simvastatin on PI3K/AKT activation, and subsequently antagonized simvastatin-induced radiosensitivity and EMT reversion. Additionally, simvastatin aggravated radiation-mediated Ec9706-R tumor growth inhibition. Together, simvastatin inhibits the development of Ec9706-R cells by increasing radiosensitivity and reversing EMT via PTEN-PI3K/AKT pathway, implying a promising strategy against EC radioresistance.     

Bmi-1 confers adaptive radioresistance to KYSE-150R esophageal carcinoma cells

Radiotherapy (RT) is a major modality of cancer treatment. However, tumors often acquire radioresistance, which causes RT to fail. The exact mechanisms by which tumor cells subjected to fractionated irradiation (FIR) develop an adaptive radioresistance are largely unknown. Using the radioresistant KYSE-150R esophageal squamous cell carcinoma (ESCC) model, which was derived from KYSE-150 parental cells using FIR, the role of Bmi-1 in mediating the radioadaptive response of ESCC cells to RT was investigated. The results showed that the level of Bmi-1 expression was significantly higher in KYSE-150R cells than in the KYSE-150 parental cells. Bmi-1 depletion sensitized the KYSE-150R cells to RT mainly through the induction of apoptosis, partly through the induction of senescence. A clonogenic cell survival assay showed that Bmi-1 depletion significantly decreased the radiation survival fraction in KYSE-150R cells. Furthermore, Bmi-1 depletion increased the generation of reactive oxygen species (ROS) and the expression of oxidase genes (Lpo, Noxo1 and Alox15) in KYSE-150R cells exposed to irradiation. DNA repair capacities assessed by γ-H2AX foci formation were also impaired in the Bmi-1 down-regulated KYSE-150R cells. These results suggest that Bmi-1 plays an important role in tumor radioadaptive resistance under FIR and may be a potent molecular target for enhancing the efficacy of fractionated RT.     

Raman Spectroscopic Study of Radioresistant Oral Cancer Sublines Established by Fractionated Ionizing Radiation

Radiotherapy is an important treatment modality for oral cancer. However, development of radioresistance is a major hurdle in the efficacy of radiotherapy in oral cancer patients. Identifying predictors of radioresistance is a challenging task and has met with little success. The aim of the present study was to explore the differential spectral profiles of the established radioresistant sublines and parental oral cancer cell lines by Raman spectroscopy. We have established radioresistant sublines namely, 50Gy-UPCI:SCC029B and 70Gy-UPCI:SCC029B from its parental UPCI:SCC029B cell line, by using clinically admissible 2Gy fractionated ionizing radiation (FIR). The developed radioresistant character was validated by clonogenic cell survival assay and known radioresistance-related protein markers like Mcl-1, Bcl-2, Cox-2 and Survivin. Altered cellular morphology with significant increase (p<0.001) in the number of filopodia in radioresistant cells with respect to parental cells was observed. The Raman spectra of parental UPCI:SCC029B, 50Gy-UPCI:SCC029B and 70Gy-UPCI:SCC029B cells were acquired and spectral features indicate possible differences in biomolecules like proteins, lipids and nucleic acids. Principal component analysis (PCA) provided three clusters corresponding to radioresistant 50Gy, 70Gy-UPCI:SCC029B sublines and parental UPCI:SCC029B cell line with minor overlap, which suggest altered molecular profile acquired by the radioresistant cells due to multiple doses of irradiation. The findings of this study support the potential of Raman spectroscopy in prediction of radioresistance and possibly contribute to better prognosis of oral cancer.     

Altered O-glycosylation is associated with inherent radioresistance and malignancy of human laryngeal carcinoma

Radioresistance (inherent or acquired) remains a major obstacle affecting the clinical outcome of radiotherapy for laryngeal carcinoma. Results from our laboratory and other groups suggest that aberrant glycosylation contributes to cancer acquired radioresistance. However, the role of glycosylation in inherent radioresistance of laryngeal carcinoma has not been fully uncovered. In this study, we investigated the glycan profiling of the inherent radioresistant (Hep-2max) and radiosensitive (Hep-2 min) cell lines using lectin microarray analysis. The results revealed that the radioresistant cell line Hep-2max presented higher core 1-type O-glycans than the sensitive one. Further analysis of the O-glycan regulation by benzyl-α-GalNAc application in Hep-2max cells showed partial inhibition of the O-glycan biosynthesis and increased radiosensitivity. In addition, core 1 β1, 3-galactosyltransferase (C1GALT1) overexpression in Hep-2 min cells enhanced cell migration, invasion, and radioresistance. Conversely, knockdown of C1GALT1 in Hep-2max cells was able to suppress these malignant phenotypes. Moreover, mechanistic investigations showed that C1GALT1 modified the O-glycans on integrin β1 and regulated its activity. The glycosylation-mediated radioresistance was further inhibited by anti-integrin β1 blocking antibody. Importantly, we also observed that core 1-type O-glycans expression was correlated with advanced tumor stage, metastasis, and poor survival of laryngeal carcinoma patients. These findings suggest that altered O-glycosylation can lead to the inherent radioresistance and progression, and therefore may be important for enhancing the efficacy of radiotherapy in laryngeal carcinoma.     

Expression of human aldo-keto reductase 1C2 in cell lines of peritoneal endometriosis: potential implications in metabolism of progesterone and dydrogesterone and inhibition by progestins

The human aldo-keto reductase AKR1C2 converts 5α-dihydrotestosterone to the less active 3α-androstanediol and has a minor 20-ketosteroid reductase activity that metabolises progesterone to 20α-hydroxyprogesterone. AKR1C2 is expressed in different peripheral tissues, but its role in uterine diseases like endometriosis has not been studied in detail. Some progestins used for treatment of endometriosis inhibit AKR1C1 and AKR1C3, with unknown effects on AKR1C2. In this study we investigated expression of AKR1C2 in the model cell lines of peritoneal endometriosis, and examined the ability of recombinant AKR1C2 to metabolise progesterone and progestin dydrogesterone, as well as its potential inhibition by progestins. AKR1C2 is expressed in epithelial and stromal endometriotic cell lines at the mRNA level. The recombinant enzyme catalyses reduction of progesterone to 20α-hydroxyprogesterone with a 10-fold lower catalytic efficiency than the major 20-ketosteroid reductase, AKR1C1. AKR1C2 also metabolises progestin dydrogesterone to its 20α-dihydrodydrogesterone, with 8.6-fold higher catalytic efficiency than 5α-dihydrotestosterone. Among the progestins that are currently used for treatment of endometriosis, dydrogesterone, medroxyprogesterone acetate and 20α-dihydrodydrogesterone act as AKR1C2 inhibitors with low μM K(i) values in vitro. Their potential in vivo effects should be further studied.     

Sulforaphane Preconditioning Sensitizes Human Colon Cancer Cells towards the Bioreductive Anticancer Prodrug PR-104A

The chemoprotective properties of sulforaphane (SF), derived from cruciferous vegetables, are widely acknowledged to arise from its potent induction of xenobiotic-metabolizing and antioxidant enzymes. However, much less is known about the impact of SF on the efficacy of cancer therapy through the modulation of drug-metabolizing enzymes. To identify proteins modulated by a low concentration of SF, we treated HT29 colon cancer cells with 2.5 μM SF. Protein abundance changes were detected by stable isotope labeling of amino acids in cell culture. Among 18 proteins found to be significantly up-regulated, aldo-keto reductase 1C3 (AKR1C3), bioactivating the DNA cross-linking prodrug PR-104A, was further characterized. Preconditioning HT29 cells with SF reduced the EC₅₀ of PR-104A 3.6-fold. The increase in PR-104A cytotoxicity was linked to AKR1C3 abundance and activity, both induced by SF in a dose-dependent manner. This effect was reproducible in a second colon cancer cell line, SW620, but not in other colon cancer cell lines where AKR1C3 abundance and activity were absent or barely detectable and could not be induced by SF. Interestingly, SF had no significant influence on PR-104A cytotoxicity in non-cancerous, immortalized human colonic epithelial cell lines expressing either low or high levels of AKR1C3. In conclusion, the enhanced response of PR-104A after preconditioning with SF was apparent only in cancer cells provided that AKR1C3 is expressed, while its expression in non-cancerous cells did not elicit such a response. Therefore, a subset of cancers may be susceptible to combined food-derived component and prodrug treatments with no harm to normal tissues.