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Myocardial ischemia/reperfusion injury (MI/RI) is an urgent problem with a great impact on health globally. However, its pathological mechanisms have not been fully elucidated. Hydroxysafflor yellow A (HSYA) has a protective effect against MI/RI. This study is aimed at further clarifying the relationship between HSYA cardioprotection and calcium overload as well as the underlying mechanisms. We verified the protective effect of HSYA on neonatal rat primary cardiomyocytes (NPCMs) and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from hypoxia-reoxygenation (HR) injury. To explore the cardioprotective mechanism of HSYA, we employed calcium fluorescence, TUNEL assay, JC-1 staining, and western blotting. Finally, cardio-ECR and patch-clamp experiments were used to explain the regulation of L-type calcium channels (LTCC) in cardioprotection mediated by HSYA. The results showed that HSYA reduced the levels of myocardial enzymes and protected NPCMs from HR injury. HSYA also restored the contractile function of hiPSC-CMs and field potential signal abnormalities caused by HR and exerted a protective effect on cardiac function. Further, we demonstrated that HSYA protects cardiomyocytes from HR injury by decreasing mitochondrial membrane potential and inhibiting apoptosis and calcium overload. Patch-clamp results revealed that MI/RI caused a sharp increase in calcium currents, which was inhibited by pretreatment with HSYA. Furthermore, we found that HSYA restored contraction amplitude, beat rate, and field potential duration of hiPSC-CMs, which were disrupted by the LTCC agonist Bay-K8644. Patch-clamp experiments also showed that HSYA inhibits Bay-K8644-induced calcium current, with an effect similar to that of the LTCC inhibitor nisoldipine. Therefore, our data suggest that HSYA targets LTCC to inhibit calcium overload and apoptosis of cardiomyocytes, thereby exerting a cardioprotective effect and reducing MI/RI injury., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2021 Jingxue Ye et al.)

Diabetic nephropathy (DN) is a serious complication of diabetes mellitus, and its prevalence has been increasing all over the world, which is also the leading cause of end-stage renal failure. Hydroxysafflor yellow A (HSYA) is the main active chemical component of Carthamus tinctorius L., and it is commonly used in patients with cardiovascular and cerebrovascular diseases in China. The aim of this study was to investigate the renal protective effects and molecular mechanisms of HSYA on high-fat diet (HFD) and streptozotocin- (STZ-) induced DN in rats. The DN rats were treated with HSYA for eight weeks. We assessed creatinine (CR), urea nitrogen (UN), glomerular volume, podocyte number, renal inflammation, oxidative stress, and cells apoptosis markers after HSYA treatment. The number of apoptotic cells was measured by the TUNEL assay, and apoptosis-related proteins BAX, caspase-3, and BCL-2 in the renal tissue were analyzed by western blot. The treatment with HSYA significantly decreased fasting blood glucose, CR, UN, and blood lipid profile, including triglyceride and total and low-density lipoprotein cholesterol, even though it did not change the rats' body weights. The western blot results indicated that HSYA reversed the upregulation of BAX and caspase-3 and significantly increased BCL-2 in renal tissue. Moreover, the levels of TNF- α and the inflammatory products, including free fatty acids (FFA) and lactic dehydrogenase (LDH) in the HSYA group, were significantly decreased. For the oxidative stress marker, the superoxide dismutase (SOD) markedly increased in the HSYA treatment group, while the malondialdehyde (MDA) in the serum and kidney tissue evidently decreased. In conclusion, HSYA treatment preserved kidney function in diabetic nephropathy in the HFD- and STZ-induced rats. The potential mechanism of renal protective effect of HSYA might be through inhibiting oxidative stress, reducing inflammatory reaction, and attenuating renal cell apoptosis. Our studies present a promising use for Hydroxysafflor yellow A in the treatment of type 2 diabetes mellitus., Competing Interests: The authors declare no conflict of interests., (Copyright © 2020 Maosheng Lee et al.)

BACKGROUND Anti-tumor properties of hydroxysafflor-yellow A (HSYA) have been recently revealed, as a series of apoptotic factors were confirmed to be regulated by HSYA and associated with peroxisome proliferator-activated receptor Gamma (PPARγ). In this study, we investigated the cell apoptosis mechanism of HSYA via activated PPARγ signal in human gastric carcinoma cells. MATERIAL AND METHODS BGC-823 cells were cultured and divided into 5 independent groups: Tumor, HSYA, HSYA+PPARγ inhibitor (GW9662), and PPARg agonist (RGZ), RGZ+GW9662. Cell proliferative activity was measured by MTT. Apoptosis and cell cycle were detected by flow cytometry. The nuclear translocation of PPARγ was detected by immunofluorescence staining chemistry, and mRNA levels of PPARγ and caspase-3 were measured by real-time qPCR. RESULTS Compared to the RGZ group, the HSYA group (100 µM) showed a similar inhibitory effect on the proliferation process of BGC-823 cells, inducing their apoptosis. As a result, the transition of BGC-823 cells from G0/G1 phase to S phase was blocked. HSYA was also found to promote the nuclear translocation of PPARγ, leading to increased expression of PPARγ and caspase-3. The regulatory effect of HSYA on BGC-823 cells could be further inhibited by PPARγ inhibitor in group GW9662. CONCLUSIONS We report the inhibitory effect of HSYA on the proliferation of BGC-823 cells, which results in activating PPARg-dependent cell cycle blocking and cell apoptosis, suggesting that PPARg is a specific type of HSYA that can induce apoptosis of BGC-823 cells.

Background: Hydroxysafflor yellow A (HSYA) is a major active component of yellow pigment extracted from safflowers; this compound possesses potent neuroprotective effects both in vitro and in vivo. However, underlying mechanism of HSYA is not fully elucidated. The present study investigated the protective effects of HSYA in rat spinal cord compression injury model and related mechanisms involved., Methods: Sprague-Dawley rats were divided as Sham, Control, and HSYA groups (n = 30 per group). Spinal cord injury (SCI) model was induced by application of vascular clips (force of 50 g, 1 min) to the dura at T9-T10 level of vertebra. Injured animals were administered with either HSYA (8 mg/kg at 1 and 6 h after injury, then 14 mg/kg, for a total of 7 days at 24-h time intervals) or equal volume of saline by intraperitoneal injection., Results: From this experiment, we discovered that SCI in rats resulted in severe trauma, which is characterized by tissue damage, lipid peroxidation, neutrophil infiltration, inflammation mediator release, and neuronal apoptosis. However, HSYA treatment significantly reduced the following: (1) degree of tissue injury (histological score) and edema; (2) neutrophil infiltration (myeloperoxidase activity); (3) oxidative stress (superoxide dismutase, malondialdehyde, and nitric oxide); (4) pro-inflammatory cytokine expression (tumor necrosis factor-α, interleukin-6, inducible nitric oxide synthase, cyclooxygenase-2); (5) nuclear factor-κB activation; (6) apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labeling staining and cysteine-aspartic protease-3 activity). Moreover, in a separate set of experiments, we clearly demonstrated that HSYA treatment significantly ameliorated recovery of limb function (as evaluated by Basso, Beattie, and Bresnahan behavioral recovery scores)., Conclusions: Treatment with HSYA restrains development of oxidative stress, inflammation response, and apoptotic events associated with SCI of rats, demonstrating that HSYA is a potential neuroprotectant for human SCI therapy.

Background: A variety of chalcones have demonstrated cytotoxic activity toward several cancer cell lines. This study aimed to investigate the cytotoxicity of four chalcones derivatives of 2-acetylthiophene in human breast cancer cell lines., Methods: MCF-7 and MDA-MB-231 cells were treated with synthesized chalcones and the cytotoxicity was evaluated by tetrazolium dye (MTT), live/dead, and DAPI assays., Results: Chalcones significantly decreased MCF-7 and MDA-MB-231 cells viability in vitro in a dose dependent manner. After 48h treatment, the IC 50 values ranging from 5.52 to 34.23μM. Chalcone 3c displayed the highest cytotoxic activity from all the tested compounds. Cytotoxic effects of compounds were confirmed in the live/dead assay. In addition, DAPI staining revealed that these compounds induce death by apoptosis., Conclusion: The data speculate that chalcone derivatives of 2-acetylthiophene may represent a source of therapeutic agents for human breast cancer., (Copyright © 2016 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

Introduction: The kava-kava plant (Piper methsyticum) is traditionally known as the pacific elixir by the pacific islanders for its role in a wide range of biological activities. The extract of the roots of this plant contains a variety of interesting molecules including Flavokawain A and this molecule is known to have anti-cancer properties. Breast cancer is still one of the leading diagnosed cancers in women today. The metastatic process is also very pertinent in the progression of tumorigenesis., Methods: MCF-7 and MDA-MB231 cells were treated with several concentrations of FKA. The apoptotic analysis was done through the MTT assay, BrdU assay, Annexin V analysis, cell cycle analysis, JC-1 mitochondrial dye, AO/PI dual staining, caspase 8/9 fluorometric assay, quantitative real time PCR and western blot. For the metastatic assays, the in vitro scratch assay, trans-well migration/invasion assay, HUVEC tube formation assay, ex vivo rat aortic ring assay, quantitative real time PCR and western blot were employed., Results: We have investigated the effects of FKA on the apoptotic and metastatic process in two breast cancer cell lines. FKA induces apoptosis in both MCF-7 and MDA-MB231 in a dose dependent manner through the intrinsic mitochondrial pathway. Additionally, FKA selectively induces a G2/M arrest in the cell cycle machinery of MDA-MB231 and G1 arrest in MCF-7. This suggests that FKA's anti-cancer activity is dependent on the p53 status. Moreover, FKA also halted the migration and invasion process in MDA-MB231. The similar effects can be seen in the inhibition of the angiogenesis process as well., Conclusions: FKA managed to induce apoptosis and inhibit the metastatic process in two breast cancer cell lines, in vitro. Overall, FKA may serve as a promising candidate in the search of a new anti-cancer drug especially in halting the metastatic process but further in vivo evidence is needed.

Coumarins and chalcones are secondary plant metabolites which have shown an array of pharmacological properties including anti-tumour activity. We have previously reported on the synthesis and anti-proliferative activity of a series of novel coumarin-chalcone hybrids. Now we report on the in vivo efficacy as well as mechanism of action of the most potent molecule of the series, S009-131. Oral administration of this molecule resulted in regression of tumours induced by HeLa cell xenografts in nod SCID mice. The molecule inhibited proliferation of cervical cancer cells (HeLa and C33A) by inducing apoptosis and arresting cell cycle at G2/M phase. Apoptosis was induced through induction of caspase-dependent intrinsic pathway and alterations in the cellular levels of Bcl-2 family proteins. The mitochondrial transmembrane potential got highly depleted in S009-131 treated cells due to an increase in Bax/Bcl-2 ratio and intracellular ROS. The molecule induced release of cytochrome c into the cytosol and activation of initiator caspase-9 and executioner caspases-3/7. Tumour suppressor protein p53 and its transcriptional target PUMA were up regulated, suggesting their role in mediating the cell death. These results suggest that S009-131 is a potent candidate for the chemotherapy of cervical carcinoma.

Objectives: Psoralea corylifolia is a plant widely used in traditional Chinese medicine, e.g. for its chemopreventive effect. To identify active substances responsible for this effect, we investigated pharmacological effects of 11 compounds isolated from the seeds of this plant (newly described substances: 7, 2', 4'-trihydroxy-3-arylcoumarin and psoracoumestan)., Methods: The influence of distinct compounds on different signal transduction pathways (cell proliferation, survival, angiogenesis and metastasis) was screened via analysis of the activity of 24 protein kinases, mitogen activated protein kinase phosphorylation via Western blot, cytotoxicity was shown using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and determination of caspase activity. Oxidative stress was detected via 2',7'-dichlorofluorescein fluorescence., Key Findings: Some compounds showed cytotoxic effects (H4IIE, Hct116, C6 cells) mainly mediated via induction of apoptosis. Distinct compounds caused a strong inhibition of MAPK/ERK kinase (MEK) phosphorylation, weak effects on extracellular-signal regulated kinase (ERK) phosphorylation and no significant effect on p38 and c-Jun amino-terminal kinase. Corylifol C and, to a lesser extent, xanthoangelol are potent protein kinase inhibitors (inhibitory concentration 50% values for epidermal growth factor receptor (EGFR): 1.1 and 4.4 × 10(-6)  μg/ml, respectively). Because EGFR, MEK and ERK are kinases involved in cellular proliferation, an inhibition of these enzymes may be useful to cause chemopreventive effects., Conclusions: Distinct compounds isolated from P. corylifolia showed a high potential to influence cellular pathways, e.g. by inhibition of protein kinases that may be interesting for pharmacological purposes., (© 2013 Royal Pharmaceutical Society.)

In this study, we investigated the effects of 24 chalcone derivatives from 2-naphthylacetophenone toward a lymphoblastic leukemia cell line (L1210). Three compounds, called R7, R13, and R15, presented concentration- and time-dependent cytotoxicity and induced cellular death by apoptosis via mitochondrial injury and oxidative stress. The effects of these compounds appear to occur through different mechanisms because R13 and R7 induced a greater disturbance of mitochondrial potential, and all compounds induced disturbances of cellular ATP content and increased caspase-3 activity before cellular death. These compounds also interfered with antioxidant enzymes activities and GSH content through different mechanisms., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Cellular damage induced by chronic inflammation is a well known cause of colon carcinogenesis. Cyclooxygenase-2 (COX-2), the enzyme that converts arachidonic acid to prostanoids, is known to play an important role in inflammation. Herbal flavonoid isoliquiritigenin (ILTG) has previously been reported to be a strong suppresser of the COX-2 pathway as well as an inducer of apoptosis. Here we report that the susceptibility to apoptosis by ILTG is dependent on the level of COX-2 in mouse colon adenocarcinoma Colon 26, which spontaneously expresses COX-2. This dependency was observed to be enhanced by blockage of the lipoxigenases (LOXs)-mediated metabolic pathway and attenuated by addition of a number of prostaglandins and thromboxanes. Taken together, these findings indicate that ILTG-induced apoptosis is negatively regulated by the COX-2 expression level.

Isoliquiritigenin (4,2',4'-trihydroxychalcone, ISL) is a natural pigment with a simple chalcone structure. In this study, we report the ISL-induced inhibition on the growth of human hepatoma cells (Hep G2) for the first time. The cell growth inhibition achieved by ISL treatment resulted in programmed cell death in a caspase activation-dependent manner, with an IC50 of 10.51 microg/mL. Outcomes of ISL treatment included the up-regulation of IkappaBalpha expression in the cytoplasm, and the decrease of NF-kappaB level as well as its activity in the nucleus. In addition, ISL also suppressed the expression of Bcl-XL and c-IAP1/2 protein, the downstream target molecule of NF-kappaB. These results demonstrated that ISL treatment inhibited the NF-kappaB cell survival-signaling pathway and induced apoptotic cell death in Hep G2 cells.

Isoliquiritigenin, which is possibly a principal anti-tumor constituent of licorice, a traditional Chinese herb, was examined for apoptosis-inducing activity in human gastric cancer MGC-803 cells. Typical morphological and biochemical features of apoptosis including cell shrinkage, chromatin condensation, DNA ladder formation, and appearance of apoptotic peaks (subG(1)) were observed in MGC-803 cells with isoliquiritigenin treatment. Using Fluo-3 and Rh123 as fluorescent probes, respectively, it was found that the intracellular free calcium concentration increased and the mitochondrial transmembrane potential (Deltapsi(m)) decreased in a dose-dependent manner in apoptotic cells. These results suggest that isoliquiritigenin induced apoptosis of MGC-803 cells through calcium- and Deltapsi(m)-dependent pathways, indicating that it is potentially useful as a natural anti-cancer agent.