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Defect in apoptotic signaling and up-regulation of drug transporters in cancer cells significantly limits the effectiveness of cancer chemotherapy. We propose that an agent inducing non-apoptotic cell death may overcome cancer drug resistance and showed that shikonin, a naturally occurring naphthoquinone, induced a cell death in MCF-7 and HEK293 distinct from apoptosis and characterized with (a) a morphology of necrotic cell death; (b) loss of plasma membrane integrity; (c) loss of mitochondrial membrane potentials; (d) activation of autophagy as a downstream consequence of cell death, but not a contributing factor; (e) elevation of reactive oxygen species with no critical roles contributing to cell death; and (f) that the cell death was prevented by a small molecule, necrostatin-1, that specifically prevents cells from necroptosis. The characteristics fully comply with those of necroptosis, a basic cell-death pathway recently identified by Degterev et al. with potential relevance to human pathology. Furthermore, we proved that shikonin showed a similar potency toward drug-sensitive cancer cell lines (MCF-7 and HEK293) and their drug-resistant lines overexpressing P-glycoprotein, Bcl-2, or Bcl-xL, which account for most of the clinical cancer drug resistance. To our best knowledge, this is the first report to document the induction of necroptosis by a small molecular compound to circumvent cancer drug resistance. [Mol Cancer Ther 2007;6(5):1641–9]

Supplementary Data from Shikonin circumvents cancer drug resistance by induction of a necroptotic death

Previous studies have identified GAPDH as a promising target for treating cancer and modulating immunity because its inhibition reduces glycolysis in cells (cancer cells and immune cells) with the Warburg effect, a modified form of cellular metabolism found in cancer cells. However, the quantitative relationship between GAPDH and the aerobic glycolysis remains unknown. Here, using siRNA-mediated knockdown of GAPDH expression and iodoacetate-dependent inhibition of enzyme activity, we examined the quantitative relationship between GAPDH activity and glycolysis rate. We found that glycolytic rates were unaffected by the reduction of GAPDH activity down to 19% ± 4.8% relative to untreated controls. However, further reduction of GAPDH activity below this level caused proportional reductions in the glycolysis rate. GAPDH knockdown or inhibition also simultaneously increased the concentration of glyceraldehyde 3-phosphate (GA3P, the substrate of GAPDH). This increased GA3P concentration countered the effect of GAPDH knockdown or inhibition and stabilized the glycolysis rate by promoting GAPDH activity. Mechanistically, the intracellular GA3P concentration is controlled by the Gibbs free energy of the reactions upstream of GAPDH. The thermodynamic state of the reactions along the glycolysis pathway was only affected when GAPDH activity was reduced below 19% ± 4.8%. Doing so moved the reactions catalyzed by GAPDH + PGK1 (phosphoglycerate kinase 1, the enzyme immediate downstream of GAPDH) away from the near-equilibrium state, revealing an important biochemical basis to interpret the rate control of glycolysis by GAPDH. Collectively, we resolved the numerical relationship between GAPDH and glycolysis in cancer cells with the Warburg effect and interpreted the underlying mechanism.

We recently reported that dibenzocyclooctadiene lignans were a novel class of P-glycoprotein (P-gp) inhibitors. In this study, we demonstrated that the lignans of this class were also effective inhibitors of multidrug resistance-associated protein 1 (MRP1). The activities of 5 dibenzocyclooctadiene lignans (schisandrin A, schisandrin B, schisantherin A, schisandrol A, and schisandrol B) to reverse MRP1-mediated drug resistance were tested using HL60/Adriamycin (ADR) and HL60/Multidrug resistance-associated protein (MRP), two human promyelocytic leukemia cell lines with overexpression of MRP1 but not P-gp. The five lignans could effectively reverse drug resistance of the two cell lines to vincristine, daunorubicin, and VP-16. This study, together with our previous reports, proves that dibenzocyclooctadiene lignans have multiple activities against cancer multidrug resistance, including inhibition of P-gp and MRP1, and enhancement of apoptosis. Considering that cancer multidrug resistance (MDR) is multifactorial, agents with broad activities are preferable to the use of combination of several specific modulators to prevent drug-drug interaction and cumulative toxicity.

Human DNA polymerases kappa, eta, iota, and zeta are responsible for the translesion DNA synthesis. Numerous in vitro studies indicated that these enzymes may contribute to DNA lesion-triggered and spontaneous mutation. We measured the transcripts of these 4 enzymes in 131 self-paired cancerous and non-tumor samples, including 23 lung cancers, 49 stomach cancers, and 59 colorectal cancers. Our results indicated that, except pol eta in colorectal cancers, these enzymes are all significantly down-regulated in human lung, stomach, and colorectal cancers, suggesting that these enzymes are probably not closely associated with the elevated mutations in human cancer.

Warburg effect is a dominant phenotype of most cancer cells. Here we show that this phenotype depends on its environment. When cancer cells are under regular culture condition, they show Warburg effect; whereas under lactic acidosis, they show a nonglycolytic phenotype, characterized by a high ratio of oxygen consumption rate over glycolytic rate, negligible lactate production and efficient incorporation of glucose carbon(s) into cellular mass. These two metabolic modes are intimately interrelated, for Warburg effect generates lactic acidosis that promotes a transition to a nonglycolytic mode. This dual metabolic nature confers growth advantage to cancer cells adapting to ever changing microenvironment.

Purpose Cancer drug resistance is a major obstacle for the success of chemotherapy. Since most clinical anticancer drugs could induce drug resistance, it is desired to develop candidate drugs that are highly efficacious but incompetent to induce drug resistance. Numerous previous studies have proven that shikonin and its analogs not only are highly tumoricidal but also can bypass drug-transporter and apoptotic defect mediated drug resistance. The purpose of this study is to investigate if or not shikonin is a weak inducer of cancer drug resistance. Experimental Design Different cell lines (K562, MCF-7, and a MDR cell line K562/Adr), after repeatedly treated with shikonin for 18 months, were assayed for drug resistance and gene expression profiling. Results After 18-month treatment, cells only developed a mere 2-fold resistance to shikonin and a marginal resistance to cisplatin and paclitaxel, without cross resistance to shikonin analogs and other anticancer agents. Gene expression profiles demonstrated that cancer cells did strongly respond to shikonin treatment but failed to effectively mobilize drug resistant machineries. Shikonin-induced weak resistance was associated with the up-regulation of βII-tubulin, which physically interacted with shikonin. Conclusion Taken together, apart from potent anticancer activity, shikonin and its analogs are weak inducers of cancer drug resistance and can circumvent cancer drug resistance. These merits make shikonin and its analogs potential candidates for cancer therapy with advantages of avoiding induction of drug resistance and bypassing existing drug resistance.

Background To mitigate the cardiotoxicity of anthracycline antibiotics without compromising their anticancer activities is still an issue to be solved. We previously demonstrated that schisandrin B (Sch B) could protect against doxorubicin (Dox)-induced acute cardiotoxicity via enhancing cardiomyocytic glutathione redox cycling that could attenuate oxidative stress generated from Dox. In this study, we attempted to prove if Sch B could also protect against Dox-induced chronic cardiotoxicity, a more clinically relevant issue, without compromising its anticancer activity. Methodology Rat was given intragastrically either vehicle or Sch B (50 mg/kg) two hours prior to i.p. Dox (2.5 mg/kg) weekly over a 5-week period with a cumulative dose of Dox 12.5 mg/kg. At the 6th and 12th week after last dosing, rats were subjected to cardiac function measurement, and left ventricles were processed for histological and ultrastructural examination. Dox anticancer activity enhanced by Sch B was evaluated by growth inhibition of 4T1, a breast cancer cell line, and S180, a sarcoma cell line, in vitro and in vivo. Principal Findings Pretreatment with Sch B significantly attenuated Dox-induced loss of cardiac function and damage of cardiomyocytic structure. Sch B substantially enhanced Dox cytotoxicities toward S180 in vitro and in vivo in mice, and increased Dox cytotoxcity against 4T1 in vitro. Although we did not observe this enhancement against the implanted 4T1 primary tumor, the spontaneous metastasis to lung was significantly reduced in combined treatment group than Dox alone group. Conclusion Sch B is capable of protecting Dox-induced chronic cardiotoxicity and enhancing its anticancer activity. To the best of our knowledge, Sch B is the only molecule ever proved to function as a cardioprotective agent as well as a chemotherapeutic sensitizer, which is potentially applicable for cancer treatment.

Purpose: The dose-cumulative cardiotoxicties and the emerging cancerous apoptotic/drug resistance are two major obstacles limiting the efficacy of anthracycline antibiotics, notably doxorubicin. We attempted to prove if schisandrin B (Sch B), a dual inhibitor of P-glycoprotein and multidrug resistance–associated protein 1, could protect against doxorubicin-induced cardiotoxicity, on the premise that Sch B is an enhancer of glutathione redox cycling that may attenuate doxorubicin-induced oxidative stress in the cardiomyocytes. Experimental Design: Mice or rat were dosed with a single injection of doxorubicin (25 mg/kg, i.p.) with or without pretreatment of Sch B. The protective roles of Sch B against doxorubicin-induced cardiac damage were evaluated on the aspects of the release of cardiac enzymes into serum, the formation of malondialdehyde, the activation of matrix metalloproteinase, the structural damage in the left ventricles, the mortality rates, and the cardiac functions. Results: Pretreatment of Sch B significantly attenuated doxorubicin-induced cardiotoxicities on all the aspects listed above. The underlying mechanism was associated with the effect of Sch B on maintaining the cardiomyocytic glutathione and the activities of superoxide dismutase, and the key enzymes (glutathione peroxidase, glutathione reductase, and glutathione transferase) responsible for glutathione redox cycling, which neutralized doxorubicin-induced oxidative stress. Conclusion: To the best of our knowledge, Sch B is the only molecule ever proved to function as a cardioprotective agent as well as a dual inhibitor of P-glycoprotein and multidrug resistance–associated protein 1, which is potentially applicable to treat cancers, especially the multidrug-resistant cancers involving doxorubicin or its kin.

Defect in apoptotic signaling and up-regulation of drug transporters in cancer cells significantly limits the effectiveness of cancer chemotherapy. We propose that an agent inducing non-apoptotic cell death may overcome cancer drug resistance and showed that shikonin, a naturally occurring naphthoquinone, induced a cell death in MCF-7 and HEK293 distinct from apoptosis and characterized with (a) a morphology of necrotic cell death; (b) loss of plasma membrane integrity; (c) loss of mitochondrial membrane potentials; (d) activation of autophagy as a downstream consequence of cell death, but not a contributing factor; (e) elevation of reactive oxygen species with no critical roles contributing to cell death; and (f) that the cell death was prevented by a small molecule, necrostatin-1, that specifically prevents cells from necroptosis. The characteristics fully comply with those of necroptosis, a basic cell-death pathway recently identified by Degterev et al. with potential relevance to human pathology. Furthermore, we proved that shikonin showed a similar potency toward drug-sensitive cancer cell lines (MCF-7 and HEK293) and their drug-resistant lines overexpressing P-glycoprotein, Bcl-2, or Bcl-xL, which account for most of the clinical cancer drug resistance. To our best knowledge, this is the first report to document the induction of necroptosis by a small molecular compound to circumvent cancer drug resistance. [Mol Cancer Ther 2007;6(5):1641–9]

A bacterial strain HLK1(T) was isolated from the human erythroleukemia cell line K562. This bacterium is a Gram-negative rod, motile with a polar flagellum. It is strictly aerobic, nonfermentative, and oxidase and catalase positive. Its optimal growth occurs at 37 degrees C at pH between 6.5 and 7.5. Phylogenetically, although it shares 98% similarity with the 16S rRNA of Phenylobacterium lituiforme, the DNA-DNA hybridization value between the two species is only 43%. HLK1(T) has a DNA G+C content of 71.2+/-0.2 mol%. It is a facultative intracellular organism and may have pathogenic relevance with humans and mammals. On the basis of the phylogenetic and phenotypic characterization, strain HLK1(T) is proposed to be classified in the genus Phenylobacterium, as P. zucineum sp. nov. The type strain is HLK1(T) (=CGMCC 1.3786(T), DSM=18354).

We recently reported that schisandrin B (Sch B) was a novel P-glycoprotein (P-gp) inhibitor. In this study, we revealed that Sch B was also an effective inhibitor of multidrug resistance-associated protein 1 (MRP1). The activities of Sch B to reverse MRP1-mediated drug resistance was tested using HL60/ADR and HL60/MRP, the human promyelocytic leukemia cell lines with the overexpression of MRP1 but not P-gp. Sch B resumed daunorubicin and carboxyfluorescein diacetate (CFDA, a specific substrate for MRP1) accumulation and retention in HL60/ADR cells in a time and concentration dependent manner. At the equimolar concentration, Sch B demonstrated significantly stronger potency than probenecid, a MRP1 inhibitor. This study, together with the previous findings, demonstrated that Sch B was a dual inhibitor of P-gp and MRP1, a type suggested to be preferable to the use of combination of two specific modulators to prevent drug-drug interaction and cumulative toxicities.

Purpose: To determine if five dibenzocyclooctadiene lingnans, a class of naturally occurring compounds from Schisandra chinensis (Turcz.) Baill, have the activities to reverse P-glycoprotein (P-gp) mediated multidrug resistance (MDR). Methods: The IC50s of four MDR cell lines (K562/Adr, MCF-7/Adr, KBv200, and Bcap37/Adr) toward daunorubicin, vincristine, and paclitaxel in the presence or absence of one of the dibenzocyclooctadiene lingnans were determined by a FACscan assay. The intracellular daunorubicin accumulation in the four MDR cell lines was determined by incubation of cells with daunorubicin (2 μg/ml) in the presence or absence of one of the dibenzocyclooctadiene lingnans by a FACscan assay. The interaction of the five dibenzocyclooctadiene lingnans with P-gp was assayed by their inhibition of 3H-azidopine photoaffinity labeling of P-gp. Results: Among the five lingnans, while schisandrin A and B, and schisantherin A demonstrated strong and comparable activities to reverse the drug resistance and the intracellular drug accumulation in four MDR cell lines, schisandrol A and B showed very limited activities. The poor activities of schisandrol A and B are possibly caused by the hydroxyl groups on the cyclooctadiene ring, because the activities of the molecules resumed when the hydroxyl group was esterified to form a benzoate. Further studies demonstrated that these compounds physically interacted with P-gp. Conclusion: Schisandrin A and B, and schisantherin A are potent P-gp inhibitor and is of potential for future clinical application.

Human DNA polymerases kappa, eta, iota, and zeta are responsible for the translesion DNA synthesis. Numerous in vitro studies indicated that these enzymes may contribute to DNA lesion-triggered and spontaneous mutation. We measured the transcripts of these 4 enzymes in 131 self-paired cancerous and non-tumor samples, including 23 lung cancers, 49 stomach cancers, and 59 colorectal cancers. Our results indicated that, except pol eta in colorectal cancers, these enzymes are all significantly down-regulated in human lung, stomach, and colorectal cancers, suggesting that these enzymes are probably not closely associated with the elevated mutations in human cancer.