Analysis of the chemical constituents and their metabolites in Orthosiphon stamineus Benth. via UHPLC-Q exactive orbitrap-HRMS and AFADESI-MSI techniques.

BACKGROUND: Known for its strong diuretic properties, the perennial herbaceous plant Orthosiphon stamineus Benth. is believed to preserve the kidney disease. This study compared the boiling water extract with powdered Orthosiphon stamineus Benth. and used a highly sensitive and high resolution UHPLC-Q-Exactive-Orbitrap-HRMS technology to evaluate its chemical composition. RESULTS: Furthermore, by monitoring the absorption of prototype components in rat plasma following oral treatment, the beneficial ingredients of the Orthosiphon stamineus Benth. decoction was discovered. Approximately 92 substances underwent a preliminary identification utilizing relevant databases, relevant literature, and reference standards. As the compound differences between the powdered Orthosiphon stamineus Benth. and its water decoction were analyzed, it was found that boiling produced additional compounds, 48 of which were new. 45 blood absorption prototype components and 49 OS metabolites were discovered from rat serum, and a kidney tissue homogenate revealed an additional 28 prototype components. Early differences in the distribution of ferulic acid, cis 4 coumaric acid, and rosmarinic acid were shown using spatial metabolomics. It was elucidated that the renal cortex region is where rosmarinic acid largely acts, offering a theoretical foundation for further studies on the application of OS in the prevention and treatment of illness as well as the preservation of kidney function. SIGNIFICANCE: In this study, UHPLC-Q Exactive Orbitrap-HRMS was employed to discern OS's chemical composition, and a rapid, sensitive, and broad-coverage AFADESI-MSI method was developed to visualize the spatial distribution of compounds in tissues.

The dysregulation of immune cells induced by uric acid: mechanisms of inflammation associated with hyperuricemia and its complications.

Changes in lifestyle induce an increase in patients with hyperuricemia (HUA), leading to gout, gouty arthritis, renal damage, and cardiovascular injury. There is a strong inflammatory response in the process of HUA, while dysregulation of immune cells, including monocytes, macrophages, and T cells, plays a crucial role in the inflammatory response. Recent studies have indicated that urate has a direct impact on immune cell populations, changes in cytokine expression, modifications in chemotaxis and differentiation, and the provocation of immune cells by intrinsic cells to cause the aforementioned conditions. Here we conducted a detailed review of the relationship among uric acid, immune response, and inflammatory status in hyperuricemia and its complications, providing new therapeutic targets and strategies.

Polymorphic USP8 allele promotes Parkinson's disease by inducing the accumulation of α-synuclein through deubiquitination.

Parkinson's disease (PD) is one of the most common neuro-degenerative diseases characterized by α-synuclein accumulation and degeneration of dopaminergic neurons. Employing genome-wide sequencing, we identified a polymorphic USP8 allele (USP8D442G) significantly enriched in Chinese PD patients. To test the involvement of this polymorphism in PD pathogenesis, we derived dopaminergic neurons (DAn) from human-induced pluripotent stem cells (hiPSCs) reprogrammed from fibroblasts of PD patients harboring USP8D442G allele and their healthy siblings. In addition, we knock-in D442G polymorphic site into the endogenous USP8 gene of human embryonic stem cells (hESCs) and derived DAn from these knock-in hESCs to explore their cellular phenotypes and molecular mechanism. We found that expression of USP8D442G in DAn induces the accumulation and abnormal subcellular localization of α-Synuclein (α-Syn). Mechanistically, we demonstrate that D442G polymorphism enhances the interaction between α-Syn and USP8 and thus increases the K63-specific deubiquitination and stability of α-Syn . We discover a pathogenic polymorphism for PD that represent a promising therapeutic and diagnostic target for PD.

Clerodendranthus spicatus inhibits epithelial-mesenchymal transition of renal tubular cells through the NF-κB/Snail signalling pathway in hyperuricaemia nephropathy.

CONTEXT: Clerodendranthus spicatus Thunb. (Labiatae) (CS), a perennial traditional Chinese medicinal herb that can reduce serum uric acid (sUA) levels and ameliorate renal function is widely used to treat hyperuricaemic nephropathy (HN). OBJECTIVE: To investigate the molecular mechanism of action of CS in HN treatment using in vivo and in vitro experiments. MATERIALS AND METHODS: Sprague-Dawley rats were randomly divided into control, HN, CS and positive control allopurinol groups. The HN group was intraperitoneally injected with 750 mg/kg oxonic acid potassium (OA), whereas the CS group was injected with OA along with a gavage of CS (low dose 3.125 g/kg, high dose 6.25 g/kg) for five weeks. For in vitro studies, uric acid-treated HK2 cells were used to verify the therapeutic mechanism of CS in HN. RESULTS: HN rats exhibit pathological phenotypes of elevated sUA levels and renal injury. CS significantly improved these symptoms and sUA (p < 0.05) and blood urea nitrogen (p < 0.01) levels, and dramatically improved renal tubular injury in HN rats. The IC50 value of UA (uric acid) in HK2 cells was 826.32 ± 3.55 µg/mL; however, 120 ng/mL CS had no significant cytotoxicity on HK2 cells. In vivo and in vitro studies showed that CS inhibited NF-κB phosphorylation and inhibited α-smooth muscle actin (α-SMA) and vimentin expression while increasing E-cadherin expression, suggesting that CS inhibited the fibrotic process in renal cells, thus protecting renal function. DISCUSSION AND CONCLUSIONS: These findings provide a fundamental understanding of the application of CS in HN treatment to better guide clinical interventions.

SLC2A9 rs16890979 reduces uric acid absorption by kidney organoids.

Introduction: The excretion and absorption of uric acid (UA) by the kidneys helps regulate serum UA levels. GLUT9, encoded by SLC2A9, is mainly expressed in the renal tubules responsible for UA absorption. SLC2A9 polymorphisms are associated with different serum UA levels. However, the lack of proper in vitro models has stalled research on the mechanisms of single nucleotide polymorphisms (SNPs) that affect UA metabolism in human urate transporters. Methods: In this study, we constructed a gene-edited human embryonic stem cells-9 (ESC-H9) derived kidney organoid bearing rs16890979, an SLC2A9 missense mutation with undetermined associations with hyperuricemia or hypouricemia. Kidney organoids derived from ESC-H9 with genetical overexpression (OE) and low expression (shRNA) of SLC2A9 to serve as controls to study the function of SLC2A9. The function of rs16890979 on UA metabolism was evaluated after placing the organoids to urate-containing medium and following histopathological analysis. Results: The kidney organoids with heterozygous or homozygous rs16890979 mutations showed normal SLC2A9 expression levels and histological distribution, phenotypically similar to the wild-type controls. However, reduced absorption of UA by the kidney organoids with rs16890979 mutants was observed. This finding together with the observation that UA absorption is increased in organoids with SLC2A9 overexpression and decreased in those with SLC2A9 knockdown, suggest that GLUT9 is responsible for UA absorption, and the rs16890979 SNP may compromise this functionality. Moreover, epithelial-mesenchymal transition (EMT) was detected in organoids after UA treatment, especially in the kidney organoid carrying GLUT9OE, suggesting the cytobiological mechanism explaining the pathological features in hyperuricosuria-related renal injury. Discussion: This study showing the transitional value of kidney organoid modeling the function of SNPs on UA metabolism. With a defined genetic background and a confirmed UA absorption function should be useful for studies on renal histological, cellular, and molecular mechanisms with this organoid model.

Corrigendum: Combination effect of three main constituents from Sarcandra glabra inhibits oxidative stress in the mice following acute lung injury: a role of MAPK-NF-κB pathway.

[This corrects the article DOI: 10.3389/fphar.2020.580064.].

Synthesis and characterization of polypyridine ruthenium(II) complexes and anticancer efficacy studies in vivo and in vitro.

In this article, ligand IPP (IPP = 4-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)-N,N-diphenylaniline) and its three Ru(II) complexes: [Ru(bpy)2(IPP)](ClO4)2 (1) (bpy = 2,2'-bipyridine), [Ru(dmbpy)2(IPP)](ClO4)2 (2) (dmbpy = 4,4'-dimethyl-2,2'-bipyridine), and [Ru(phen)2(IPP)](ClO4)2 (3) (phen = 1,10-phenanthroline) were synthesized and characterized. The anticancer activity in vitro of the complexes was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The scratching and colony-forming experiments confirmed the complexes 1, 2, 3 interfered with the proliferation and migration ability of cells. The accumulation of the complexes in cells was researched and we found that these complexes directly accumulated in mitochondria, then the complexes cause a decline of the mitochondrial membrane potential and induce an increase of intracellular reactive oxygen species (ROS) levels. The growth of B16 cells were inhibited by 1, 2 and 3 at G0/G1 phase. Apoptosis was induced through mitochondrial pathway and the expression of apoptosis-related factors was regulated. In addition, the complexes promoted the transition of poly(ADP-ribose)polymerase (PARP) into the cleaved form (Cleaved PARP), downregulated the anti-apoptotic proteins, and upregulated the pro-apoptotic proteins. Consequently, complexes 1, 2 and 3 exerted their anticancer activity by regulating B-cell lymphoma-2 (Bcl-2) family proteins. Complex 2 showed excellent antitumor effects with a high inhibitory rate of 65.95% in vivo. Taken together, the complexes cause apoptosis in B16 cells through a ROS-mediated mitochondrial dysfunction pathway.

Two Sets of Compound Complex Driving for High Efficiency of Nonintegration Reprogramming of Human Fibroblasts.

Currently, plentiful chemical-assisted methods have been applied for mouse induced pluripotent stem cells (iPSCs). It has been reported that small-molecule compounds can only reprogram mouse embryonic fibroblasts into mouse chemically induced pluripotent stem cells (mouse CiPSCs). However, human CiPSCs have not been reported. Therefore, it is still necessary to search for safer chemically assisted human pluripotent stem cells, which might realize the potential of human iPSCs. Here, we developed two sets of chemical cocktails to greatly improve the induction efficiency of human nonintegrated iPSCs, including the 4 compound mixture (4M) and the 5 compound mixture (4MI). These two sets of complex driving strategies might greatly improve the reprogramming efficiency to generate integration-free iPSCs.

Genetically Predicted Telomere Length and Its Relationship With Alzheimer's Disease.

Are shorter telomeres causal risk factors for Alzheimer's disease (AD)? This study aimed to examine if shorter telomeres were causally associated with a higher risk of AD using Mendelian randomization (MR) analysis. Two-sample MR methods were applied to the summary effect sizes and standard errors from a genome-wide association study for AD. Twenty single nucleotide polymorphisms of genome-wide significance were selected as instrumental variables for leukocyte telomere length. The main analyses were performed primarily using the random-effects inverse-variance weighted method and complemented with the other three methods: weighted median approaches, MR-Egger regression, and weighted mode approach. The intercept of MR-Egger regression was used to assess horizontal pleiotropy. We found that longer telomeres were associated with lower risks of AD (odds ratio = 0.79, 95% confidence interval: 0.67, 0.93, P = 0.004). Comparable results were obtained using weighted median approaches, MR-Egger regression, and weighted mode approaches. The intercept of the MR-Egger regression was close to zero. This may show that there was not suggestive of horizontal pleiotropy. Our findings provided additional evidence regarding the putative causal association between shorter telomere length and the higher risk of AD.

Combination Effect of Three Main Constituents From Sarcandra glabra Inhibits Oxidative Stress in the Mice Following Acute Lung Injury: A Role of MAPK-NF-κB Pathway.

Caffeoylquinic acids, coumarins and dicaffeoyl derivatives are considered to be three kinds of the most abundant bioactive components in Sarcandra glabra, an anti-inflammatory herb mainly found in Southern Asia. The combined anti-inflammatory effect of three typical constituents C + R + I (chlorogenic acid + rosmarinic acid + isofraxidin) from this plant has been investigated. The result implies that targeting the MAPK-NF-κB pathway would be one of the major mechanisms involved, using LPS stimulated RAW 264.7 cells as in vitro model and LPS-induced acute lung injury in mice as in vivo model. C + R + I can significantly suppress the levels of nitric oxide (NO), pro-inflammatory cytokines, and inhibit iNOS and COX-2 expression in LPS-treated RAW264.7 macrophage cells. Western blot analysis showed that C + R + I suppressed phosphorylation of NF-κB and MAPK, including phosphorylation of p65-NF-κB, IKB, ERK, JNK and P38. Besides, C + R + I suppressed MPO protein expression, but promoted SOD and HO-1 expression, and the related targets for C, R, and I were also predicted by molecular docking. This indicated that C + R + I could alleviate oxidative stress induced by LPS, which were further verified in the in vivo model of mice with acute lung injury through the measurement of corresponding inflammatory mediators and the analysis of immunehistochemistry.

Functions of p53 in pluripotent stem cells.

Pluripotent stem cells (PSCs) are capable of unlimited self-renewal in culture and differentiation into all functional cell types in the body, and thus hold great promise for regenerative medicine. To achieve their clinical potential, it is critical for PSCs to maintain genomic stability during the extended proliferation. The critical tumor suppressor p53 is required to maintain genomic stability of mammalian cells. In response to DNA damage or oncogenic stress, p53 plays multiple roles in maintaining genomic stability of somatic cells by inducing cell cycle arrest, apoptosis, and senescence to prevent the passage of genetic mutations to the daughter cells. p53 is also required to maintain the genomic stability of PSCs. However, in response to the genotoxic stresses, a primary role of p53 in PSCs is to induce the differentiation of PSCs and inhibit pluripotency, providing mechanisms to maintain the genomic stability of the self-renewing PSCs. In addition, the roles of p53 in cellular metabolism might also contribute to genomic stability of PSCs by limiting oxidative stress. In summary, the elucidation of the roles of p53 in PSCs will be a prerequisite for developing safe PSC-based cell therapy.

Neuroprotective Effect of Optimized Yinxieling Formula in 6-OHDA-Induced Chronic Model of Parkinson's Disease through the Inflammation Pathway.

Parkinson's disease (PD) is characterized by progressive degeneration of dopaminergic neurons in the substantia nigra (SN)-striatum circuit, which is associated with glial activation and consequent chronic neuroinflammation. Optimized Yinxieling Formula (OYF) is a Chinese medicine that exerts therapeutical effect and antiinflammation property on psoriasis. Our previous study has proven that pretreatment with OYF could regulate glia-mediated inflammation in an acute mouse model of PD induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Given that PD is a chronic degeneration disorder, this study applied another PD animal model induced by striatal injection of 6-hydroxydopamine (6-OHDA) to mimic the progressive damage of the SN-striatum dopamine system in rats. The OYF was administrated in the manner of pretreatment plus treatment. The effects of the OYF on motor behaviors were assessed with the apomorphine-induced rotation test and adjusting steps test. To confirm the effect of OYF on dopaminergic neurons and glia activation in this model, we analyzed the expression of tyrosine hydroxylase (TH) and glia markers, ionized calcium-binding adapter molecule 1 (Iba-1), and glial fibrillary acidic protein (GFAP) in the SN region of the rat PD model. Inflammation-associated factors, including tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), IL-6, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), were further evaluated in this model and in interferon-γ- (INF-γ-) induced murine macrophages RAW264.7 cells. The results from the in vivo study showed that OYF reversed the motor behavioral dysfunction in 6-OHDA-induced PD rats, upregulated the TH expression, decreased the immunoreactivity of Iba-1 and GFAP, and downregulated the mRNA levels of TNF-α and COX-2. The OYF also trended to decrease the mRNA levels of IL-1ß and iNOS in vivo. The results from the in vitro study showed that OYF significantly decreased the mRNA levels of TNF-α, IL-1ß, IL-6, iNOS, and COX-2. Therefore, this study suggests that OYF exerts antiinflammatory effects, which might be related to the protection of dopaminergic neurons in 6-OHDA-induced chronic neurotoxicity.

Studies of the structure-antioxidant activity relationships and antioxidant activity mechanism of iridoid valepotriates and their degradation products.

Oxidative stress has been associated with diverse diseases, including obesity, cancer and neurodegeneration. In fact, Valeriana jatamansi Jones (valerian) and its extracts possess strong antioxidant activities that extend their application in clinical practice to the treatment of these illnesses, even though the underlying mechanisms are not well understood. Iridoid valepotriate, a characteristic iridoid ester in valerian with poor chemical stability, possesses considerable antioxidant components. The original compounds and their degradation products have been found to exhibit strong antioxidant activities. However, the relationship between their structure and antioxidant effects and the mechanism underlying their oxidation resistance remain unclear. A forced degradation study using three iridoid valepotriates (valtrate, acevaltrate and 1-ß acevaltrate) was performed in this work, and the structures of their degradation products were estimated by TLC-MS and LC-MS. Comparison of the antioxidant activities of the iridoid valepotriates before and after forced degradation revealed that degradation reduced the activities of the iridoid valepotriates in free radical scavenging and cytotoxic and cell apoptosis tests. The results suggested that the oxirane nucleus is important for defining the antioxidant profile of iridoid valepotriate. We uncovered possible mechanisms that could explain the antioxidant activities, including the generation of two hydroxyl groups through intramolecular transfer of an H• from an oxirane ring and a reduction in ROS levels through interactions with GABAergic signalling pathways.

Ursolic acid sensitizes cisplatin-resistant HepG2/DDP cells to cisplatin via inhibiting Nrf2/ARE pathway.

BACKGROUND: Combinations of adjuvant sensitizers with anticancer drugs is a promising new strategy to reverse chemoresistance. Ursolic acid (UA) is one of the natural pentacyclic triterpene compounds known to have many pharmacological characteristics such as anti-inflammatory and anticancer properties. This study investigates whether UA can sensitize hepatocellular carcinoma cells to cisplatin. MATERIALS AND METHODS: Cells were transfected with nuclear factor erythroid-2-related factor 2 (Nrf2) small interfering RNA and Nrf2 complementary DNA by using Lipofectin 2000. The cytotoxicity of cells was investigated by Cell Counting Kit 8 assay. Cell apoptosis, cell cycle, reactive oxygen species, and mitochondrial membrane potential were detected by flow cytometry fluorescence-activated cell sorting. The protein level of Nrf2, NAD(P)H quinone oxidoreductase 1 (NQO1), glutathione S-transferase (GST), and heme oxygenase-1 (HO-1) was detected by Western blot analysis. RESULTS: The results showed that the reverse index was 2.9- and 9.69-fold by UA of 1.125 µg/mL and 2.25 µg/mL, respectively, for cisplatin to HepG2/DDP cells. UA-cisplatin combination induced cell apoptosis and reactive oxygen species, blocked the cell cycle in G0/G1 phase, and reduced the mitochondrial membrane potential. Mechanistically, UA-cisplatin dramatically decreased the expression of Nrf2 and its downstream genes. The sensibilization of UA-cisplatin combination was diminished in Nrf2 small interfering RNA-transfected HepG2/DDP cells, as well as in Nrf2 complementary DNA-transfected HepG2/DDP cells. CONCLUSION: The results confirmed the sensibilization of UA on HepG2/DDP cells to cisplatin, which was possibly mediated via the Nrf2/antioxidant response element pathway.

Artemisitene activates the Nrf2-dependent antioxidant response and protects against bleomycin-induced lung injury.

The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is a crucial regulator of the cellular antioxidant response and xenobiotic metabolism. Activation of the Nrf2 signaling pathway has been demonstrated to confer protection against environmental insults and prevent disease or inhibit the progression of diseases related to oxidative stress. In an attempt to identify novel improved Nrf2 inducers for systemic protection against tissue damage by environmental insults, we identified artemisitene as a novel Nrf2 activator using antioxidant responsive element luciferase assay in MDA-MB-231 cells. Further studies suggest that artemisitene activates Nrf2 by decreasing Nrf2 ubiquitination and increasing its stability. In Nrf2 wild-type mice, systemic administration of artemisitene strongly inhibits bleomycin-induced lung damage. Artemisitene represents a novel class of Nrf2 inducer, and artemisitene-based therapeutic approach targeting Nrf2 may also provide antioxidant protection for humans against tissue damage by toxic chemicals.-Chen, W., Li, S., Li, J., Zhou, W., Wu, S., Xu, S., Cui, K., Zhang, D. D., Liu, B. Artemisitene activates the Nrf2-dependent antioxidant response and protects against bleomycin-induced lung injury.

Reprogramming with Small Molecules instead of Exogenous Transcription Factors.

Induced pluripotent stem cells (iPSCs) could be employed in the creation of patient-specific stem cells, which could subsequently be used in various basic and clinical applications. However, current iPSC methodologies present significant hidden risks with respect to genetic mutations and abnormal expression which are a barrier in realizing the full potential of iPSCs. A chemical approach is thought to be a promising strategy for safety and efficiency of iPSC generation. Many small molecules have been identified that can be used in place of exogenous transcription factors and significantly improve iPSC reprogramming efficiency and quality. Recent studies have shown that the use of small molecules results in the generation of chemically induced pluripotent stem cells from mouse embryonic fibroblast cells. These studies might lead to new areas of stem cell research and medical applications, not only human iPSC by chemicals alone, but also safe generation of somatic stem cells for cell based clinical trials and other researches. In this paper, we have reviewed the recent advances in small molecule approaches for the generation of iPSCs.

A naturally occurring CD8(+)CD122(+) T-cell subset as a memory-like Treg family.

Despite extensive studies on CD4(+)CD25(+) regulatory T cells (Tregs) during the past decade, the progress on their clinical translation remains stagnant. Mounting evidence suggests that naturally occurring CD8(+)CD122(+) T cells are also Tregs with the capacity to inhibit T-cell responses and suppress autoimmunity as well as alloimmunity. In fact, they are memory-like Tregs that resemble a central memory T cell (TCM) phenotype. The mechanisms underlying their suppression are still not well understood, although they may include IL-10 production. We have recently demonstrated that programmed death-1 (PD-1) expression distinguishes between regulatory and memory CD8(+)CD122(+) T cells and that CD8(+)CD122(+) Tregs undergo faster homeostatic proliferation and are more potent in the suppression of allograft rejection than conventional CD4(+)CD25(+) Tregs. These findings may open a new line of investigation for accelerating effective Treg therapies in the clinic. In this review, we summarize the significant progress in this promising field of CD8(+)CD122(+) Treg research and discuss their phenotypes, suppressive roles in autoimmunity and alloimmunity, functional requirements, mechanisms of action and potential applications in the clinic.

Synthesis, structures, cellular uptake and apoptosis-inducing properties of highly cytotoxic ruthenium-Norharman complexes.

Three novel Ru(II) complexes of the general formula [Ru(N-N)(2)(Norharman)(2)](SO(3)CF(3))(2), where N-N = 2,2'-bipyridine (bpy, 1), 1,10-phenanthroline (phen, 2), 4,7-diphenyl-1,10-phenanthroline (DIP, 3) and Norharman (9H-pyrido[3,4-b]indole) is a naturally occurring ß-carboline alkaloid, have been synthesized and characterized. The molecular structures of 1 and 2 have been determined by X-ray diffraction analysis. The cellular uptake efficiencies, in vitro cytotoxicities and apoptosis-inducing properties of these complexes have been extensively explored. Notably, 1-3 exhibit potent antiproliferative activities against a panel of human cancer cell lines with IC(50) values lower than those of cisplatin. Further studies show that 1-3 can cause cell cycle arrest in the G0/G1 phase and induce apoptosis through mitochondrial dysfunction and reactive oxygen species (ROS) generation. In vitro DNA binding studies have also been conducted to provide information about the possible mechanism of action.

Nuclear permeable ruthenium(II) ß-carboline complexes induce autophagy to antagonize mitochondrial-mediated apoptosis.

The role of autophagy in cancer development and response to cancer therapy has been a subject of debate. Here we demonstrate that a series of ruthenium(II) complexes containing a ß-carboline alkaloid as ligand can simultaneously induce autophagy and apoptosis in tumor cells. These Ru(II) complexes are nuclear permeable and highly active against a panel of human cancer cell lines, with complex 3 displaying activities greater than those of cisplatin. The antiproliferative potentialities of 1-3 are in accordance with their relative lipophilicities, cell membrane penetration abilities, and in vitro DNA binding affinities. Complexes 1-3 trigger release of reactive oxygen species (ROS) and attenuation of ROS by scavengers reduced the sub-G1 population, suggesting ROS-dependent apoptosis. Inhibition of ROS generation also reduces autophagy, indicating that ROS triggers autophagy. Further studies show that suppression of autophagy using pharmacological inhibitors (3-methyladenine and chloroquine) enhances apoptotic cell death.