EGFR mutation mediates resistance to EGFR tyrosine kinase inhibitors in NSCLC: From molecular mechanisms to clinical research.

With the development of precision medicine, molecular targeted therapy has been widely used in the field of cancer, especially in non-small-cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR) is a well-recognized and effective target for NSCLC therapies, targeted EGFR therapy with EGFR-tyrosine kinase inhibitors (EGFR-TKIs) has achieved ideal clinical efficacy in recent years. Unfortunately, resistance to EGFR-TKIs inevitably occurs due to various mechanisms after a period of therapy. EGFR mutations, such as T790M and C797S, are the most common mechanism of EGFR-TKI resistance. Here, we discuss the mechanisms of EGFR-TKIs resistance induced by secondary EGFR mutations, highlight the development of targeted drugs to overcome EGFR mutation-mediated resistance, and predict the promising directions for development of novel candidates.

Design, synthesis and biological evaluation of resveratrol-cinnamoyl derivates as tubulin polymerization inhibitors targeting the colchicine binding site.

A novel series of resveratrol-cinnamoyl hybrids as tubulin polymerization inhibitors were designed and synthesized, and evaluated for their anti-proliferative activities against A549, MCF-7, HepG2, HeLa and MDA-MB-231 five cancer cell lines. Most designed compounds showed better anti-proliferative activities. Particularly, compound 6h exhibited the potent anti-proliferative activities with the IC50 value of 0.12, 0.016, 0.44, 0.37 and 0.78 µΜ against A549, MCF-7, HepG2, HeLa and MDA-231, respectively, which was superior to that of reference drug colchicine. Besides, compound 6h displayed a remarkable inhibition of tubulin polymerization and a great potency to compete with [3H] colchicine in binding to tubulin. Further studies indicated that compound 6h could induce the MCF-7 cells arrest in the G2/M phase. What' more, compound 6h induced cell apoptosis in a dose-dependent manner, and regulated the expression level of apoptosis-related proteins. These results revealed that compound 6h is a promising tubulin polymerization inhibitor for treatment of cancer and it is worthy of further exploitation.

Sanguinarine induces apoptosis in osteosarcoma by attenuating the binding of STAT3 to the single-stranded DNA-binding protein 1 (SSBP1) promoter region.

BACKGROUND AND PURPOSE: Osteosarcoma, a primary malignant bone tumour prevalent among adolescents and young adults, remains a considerable challenge despite protracted progress made in enhancing patient survival rates over the last 40 years. Consequently, the development of novel therapeutic approaches for osteosarcoma is imperative. Sanguinarine (SNG), a compound with demonstrated potent anticancer properties against various malignancies, presents a promising avenue for exploration. Nevertheless, the intricate molecular mechanisms underpinning SNG's actions in osteosarcoma remain elusive, necessitating further elucidation. EXPERIMENTAL APPROACH: Single-stranded DNA-binding protein 1 (SSBP1) was screened out by differential proteomic analysis. Apoptosis, cell cycle, reactive oxygen species (ROS) and mitochondrial changes were assessed via flow cytometry. Western blotting and quantitative real-time reverse transcription PCR (qRT-PCR) were used to determine protein and gene levels. The antitumour mechanism of SNG was explored at a molecular level using chromatin immunoprecipitation (ChIP) and dual luciferase reporter plasmids. KEY RESULTS: Our investigation revealed that SNG exerted an up-regulated effect on SSBP1, disrupting mitochondrial function and inducing apoptosis. In-depth analysis uncovered a mechanism whereby SNG hindered the JAK/signal transducer and activator of transcription 3 (STAT3) signalling pathway, relieved the inhibitory effect of STAT3 on SSBP1 transcription, and inhibited the downstream PI3K/Akt/mTOR signalling axis, ultimately activating apoptosis. CONCLUSIONS AND IMPLICATIONS: The study delved further into elucidating the anticancer mechanism of SNG in osteosarcoma. Notably, we unravelled the previously undisclosed apoptotic potential of SSBP1 in osteosarcoma cells. This finding holds substantial promise in advancing the development of novel anticancer drugs and identification of therapeutic targets.

Discovery of a potent inhibitor of chaperone-mediated autophagy that targets the HSC70-LAMP2A interaction in non-small cell lung cancer cells.

BACKGROUND AND PURPOSE: Chaperone-mediated autophagy (CMA) is a selective type of autophagy targeting protein degradation and maintains high activity in many malignancies. Inhibition of the combination of HSC70 and LAMP2A can potently block CMA. At present, knockdown of LAMP2A remains the most specific method for inhibiting CMA and chemical inhibitors against CMA have not yet been discovered. EXPERIMENTAL APPROACH: Levels of CMA in non-small cell lung cancer (NSCLC) tissue samples were confirmed by tyramide signal amplification dual immunofluorescence assay. High-content screening was performed based on CMA activity, to identify potential inhibitors of CMA. Inhibitor targets were determined by drug affinity responsive target stability-mass spectrum and confirmed by protein mass spectrometry. CMA was inhibited and activated to elucidate the molecular mechanism of the CMA inhibitor. KEY RESULTS: Suppression of interactions between HSC70 and LAMP2A blocked CMA in NSCLC, restraining tumour growth. Polyphyllin D (PPD) was identified as a targeted CMA small-molecule inhibitor through disrupting HSC70-LAMP2A interactions. The binding sites for PPD were E129 and T278 at the nucleotide-binding domain of HSC70 and C-terminal of LAMP2A, respectively. PPD accelerated unfolded protein generation to induce reactive oxygen species (ROS) accumulation by inhibiting HSC70-LAMP2A-eIF2α signalling axis. Also, PPD prevented regulatory compensation of macroautophagy induced by CMA inhibition via blocking the STX17-SNAP29-VAMP8 signalling axis. CONCLUSIONS AND IMPLICATIONS: PPD is a targeted CMA inhibitor that blocked both HSC70-LAMP2A interactions and LAMP2A homo-multimerization. CMA suppression without increasing the regulatory compensation from macroautophagy is a good strategy for NSCLC therapy.

Structural insight into the bulge-containing KRAS oncogene promoter G-quadruplex bound to berberine and coptisine.

KRAS is one of the most highly mutated oncoproteins, which is overexpressed in various human cancers and implicated in poor survival. The G-quadruplex formed in KRAS oncogene promoter (KRAS-G4) is a transcriptional modulator and amenable to small molecule targeting. However, no available KRAS-G4-ligand complex structure has yet been determined, which seriously hinders the structure-based rational design of KRAS-G4 targeting drugs. In this study, we report the NMR solution structures of a bulge-containing KRAS-G4 bound to berberine and coptisine, respectively. The determined complex structure shows a 2:1 binding stoichiometry with each compound recruiting the adjacent flacking adenine residue to form a "quasi-triad plane" that stacks over the two external G-tetrads. The binding involves both π-stacking and electrostatic interactions. Moreover, berberine and coptisine significantly lowered the KRAS mRNA levels in cancer cells. Our study thus provides molecular details of ligand interactions with KRAS-G4 and is beneficial for the design of specific KRAS-G4-interactive drugs.

Single-cell sequencing reveals the landscape of the tumor microenvironment in a skeletal undifferentiated pleomorphic sarcoma patient.

Skeletal undifferentiated pleomorphic sarcoma (SUPS) is an invasive pleomorphic soft tissue sarcoma with a high degree of malignancy and poor prognosis. It is prone to recur and metastasize. The tumor microenvironment (TME) and the pathophysiology of SUPS are barely described. Single-cell RNA sequencing (scRNA-seq) provides an opportunity to dissect the landscape of human diseases at an unprecedented resolution, particularly in diseases lacking animal models, such as SUPS. We performed scRNA-seq to analyze tumor tissues and paracancer tissues from a SUPS patient. We identified the cell types and the corresponding marker genes in this SUPS case. We further showed that CD8+ exhausted T cells and Tregs highly expressed PDCD1, CTLA4 and TIGIT. Thus, PDCD1, CTLA4 and TIGIT were identified as potential targets in this case. We applied copy number karyotyping of aneuploid tumors (CopyKAT) to distinguish malignant cells from normal cells in fibroblasts. Our study identified eight malignant fibroblast subsets in SUPS with distinct gene expression profiles. C1-malignant Fibroblast and C6-malignant Fibroblast in the TME play crucial roles in tumor growth, angiogenesis, metastasis and immune response. Hence, targeting malignant fibroblasts could represent a potential strategy for this SUPS therapy. Intervention via tirelizumab enabled disease control, and immune checkpoint inhibitors (ICIs) of PD-1 may be considered as the first-line option in patients with SUPS. Taken together, scRNA-seq analyses provided a powerful basis for this SUPS treatment, improved our understanding of complex human diseases, and may afforded an alternative approach for personalized medicine in the future.

Polyphyllin I promotes cell death via suppressing UPR-mediated CHOP ubiquitination and degradation in non-small cell lung cancer.

Polyphyllin I (PPI) purified from Polyphyllarhizomes displays puissant cytotoxicity in many kinds of cancers. Several researches investigated its anti-cancer activity. But novel mechanisms are still worth investigation. This study aimed to explore PPI-induced endoplasmic reticulum (ER) stress as well as the underlying mechanism in non-small cell lung cancer (NSCLC). Cell viability or colony-forming was detected by MTT or crystal violet respectively. Cell cycle, apoptosis, reactive oxygen species (ROS) and mitochondrial membrane potential were assessed by flow cytometry. Gene and protein levels were evaluated by qRT-PCR and immunoblotting respectively. Protein interaction was determined by immunoprecipitation or immunofluorescence assay. Gene overexpression or silencing was carried out by transient transfection with plasmids or small interfering RNAs. The Cancer Genome Atlas (TCGA) database was used for Gene Set Enrichment Analysis (GSEA), survival analysis, gene expression statistics or pathway enrichment assay. PPI inhibited the propagation of NSCLC cells, increased non-viable apoptotic cells, arrested cell cycle at G2/M phase, induced ROS levels but failed to decrease mitochondrial membrane potential. High levels of GRP78 indicates poor prognosis in NSCLC patients. PPI selectively suppressed unfolded protein response (UPR)-induced GRP78 expression, subsequently protected CHOP from GRP78-mediated ubiquitination and degradation. We demonstrated that the natural product PPI, obtained from traditional herbal medicine, deserves for further study as a valuable candidate for lead compound in the chemotherapy of NSCLC.

The Chk2-PKM2 axis promotes metabolic control of vasculogenic mimicry formation in p53-mutated triple-negative breast cancer.

Vasculogenic mimicry (VM) formation, which participates in the process of neovascularization, is highly activated in p53-mutated triple-negative breast cancer (TNBC). Here, we show that Chk2 is negatively correlated with VM formation in p53-mutated TNBC. Its activation by DNA-damaging agents such as cisplatin, etoposide, and DPT reduces VM formation. Mechanistically, the Chk2-PKM2 axis plays an important role in the inhibition of VM formation at the level of metabolic regulation. Chk2 promotes the Chk2-PKM2 interaction through the Chk2 SCD (SQ/TQ cluster domain) and the PKM2 C domain. Furthermore, Chk2 promotes the nuclear export of PKM2 by phosphorylating PKM2 at Ser100. P-PKM2 S100 reduces VM formation by decreasing glucose flux, and the PKM2 S100A mutation abolishes the inhibition of glucose flux and VM formation induced by Chk2 activation. Overall, this study proposes a novel strategy of VM suppression through Chk2 induction, which prevents PKM2-mediated glucose flux in p53-mutated TNBC.

Vielanin K enhances doxorubicin-induced apoptosis via activation of IRE1α- TRAF2 - JNK pathway and increases mitochondrial Ca2 + influx in MCF-7 and MCF-7/MDR cells.

BACKGROUND: Therapeutic failure and drug resistance are common and have important implications in the poor prognosis of advanced breast cancer. It is necessary to acquire a natural product to overcome the resistance of cancer and increase the sensitivity of drug-resistant cells to anticancer agents. PURPOSE: To demonstrate whether the compound Vielanin K (VK) has the potential to increase the sensitivity of MCF-7 and MCF-7/MDR cells to anticancer agents. METHODS: Cell viability and proliferative capacity were determined by MTT, colony formation and EdU assays. Apoptosis and Ca2+ accumulation were evaluated by flow cytometry. Then, proteins were detected by immunoblotting, and gene expression levels were explored by qRT-PCR. RESULTS: In MCF-7 and corresponding MDR cells, VK increased the fluorescence intensity of Rho123, but not CFDA. VK treatment did not affect the protein expression of P-gp, MRP1 or BCRP. VK treatment enhanced the DOX-induced apoptotic cascade, while VK combined with DOX increased JNK phosphorylation by activating the IRE1α-TRAF2 signaling pathway. In addition, Ca2+ was released from the endoplasmic reticulum following combination treatment, thereby giving rise to mitochondrial apoptosis. Silencing IRE1α and JNK with small interfering RNA (siRNA) efficiently attenuated combination treatment-induced apoptosis. These effects caused mitochondrial depolarization and reduced viability in MCF-7 and corresponding MCF-7/MDR cells. CONCLUSION: VK combined with DOX increases the apoptosis of MCF-7 and corresponding MCF-7/MDR cells by activating ER stress and mitochondrial apoptosis via IRE1α-TRAF2-JNK signaling.

Synthesis and biological evaluation of novel shikonin-benzo[b]furan derivatives as tubulin polymerization inhibitors targeting the colchicine binding site.

A novel series of shikonin-benzo[b]furan derivatives were designed and synthesized as tubulin polymerization inhibitors, and their biological activities were evaluated. Most compounds revealed the comparable anti-proliferation activities against the cancer cell lines to that of shikonin and simultaneously low cytotoxicity to non-cancer cells. Among them, compound 6c displayed powerful anti-cancer activity with the IC50 value of 0.18 µM against HT29 cells, which was significantly better than that of the reference drugs shikonin and CA-4. What's more, 6c could inhibit tubulin polymerization and compete with [3H] colchicine in binding to tubulin. Further biological studies depicted that 6c can induce cell apoptosis and cell mitochondria depolarize, regulate the expression of apoptosis related proteins in HT29 cells. Besides, 6c actuated the HT29 cell cycle arrest at G2/M phase, and influenced the expression of the cell-cycle related protein. Moreover, 6c displayed potent inhibition on cell migration and tube formation that contributes to the antiangiogenesis. These results prompt us to consider 6c as a potential tubulin polymerization inhibitor and is worthy for further study.

MicroRNA and long noncoding RNA involvement in gout and prospects for treatment.

MicroRNAs (miRNAs) and long noncoding RNAs (lncRNAs) are both types of noncoding RNA. They have been demonstrated to be involved in the regulation of various human inflammatory diseases and can be used as biomarkers for disease diagnosis and prognosis, and even be developed into new drugs. Gout is an arthritic disease caused by the deposition of monosodium urate crystal (MSU) in the joints, which can lead to acute inflammation and damage adjacent tissue. Recent studies have shown that miRNAs and lncRNAs mediate the progress of gout. Based on the pathogenesis of gout, including hyperuricemia, MSU deposition, acute gouty arthritis and gouty bone erosion, this paper reviewed the role of miRNAs and lncRNAs in the processes and the possible therapeutic targets of miRNAs and lncRNAs in gout.

Toonasindiynes A-F, new polyacetylenes from Toona sinensis with cytotoxic and anti-inflammatory activities.

The plants of genus Toona are well known for diverse limonoid secondary metabolites, while polyacetylenes are rarely found from Toona species. In this work, six new polyacetylenes toonasindiynes A-F (1-6) and six known analogues (7-12) were isolated from the root bark of Toona sinensis. Their structures and absolute configurations were elucidated by HRESIMS, 1D and 2D NMR spectroscopic analysis, modified Mosher's method, and biosynthetic consideration. These polyacetylenes share the same 4,6-diyne moiety with different side chain length and different oxidation degree. Bioactivity screening revealed the cytotoxic activity of 3, 5, 9, and 11 against U2OS cells, and the inhibitory effects on nitric oxide (NO) production of 1, 2, 5, 8, 9, and 11 in lipopolysaccharide-induced RAW 264.7 cells.

Upregulation of RAGE at the blood-brain barrier in streptozotocin-induced diabetic mice.

Deposition of amyloid-beta peptide (Abeta) in the brain of diabetes is poorly understood. The receptor for advanced glycation end products (RAGE) at the blood-brain barrier (BBB) is critical for regulation of Abeta homeostasis in the brain. In this studies, we used streptozotocin-induced diabetic mice to observe the expression of RAGE at the BBB by Western blot and immunocytochemical analysis, and the in vivo blood-to-brain influx transport of (125)I-Abeta(1-) (40) using the permeability surface area product (PS) and brain capillary uptake. In the diabetic mice with hyperglycemia (>16.0 mmol/L) at 6 weeks, RAGE expression at the BBB was significantly upregulated, no significant changes of RAGE levels were found at 1 and 3 weeks after diabetes induction. The data of PS and brain capillary uptake for Abeta showed significant RAGE-dependent transport of Abeta across the BBB and substantial RAGE-dependent brain capillary uptake at 6 weeks after diabetes induction. We conclude that the upregulation of RAGE at the BBB contributes to cerebral Abeta deposition in the diabetes.

Glutathione depletion upregulates P-glycoprotein expression at the blood-brain barrier in rats.

OBJECTIVES: Glutathione (GSH) depletion has been implicated in the pathogenesis of neurological diseases. During GSH depletion, cells of the blood-brain barrier are subjected to chronic oxidative stress. Using an in-vivo system, we have investigated whether glutathione depletion changed expression of P-glycoprotein at the blood-brain barrier in rats. METHODS: Diethyl maleate was intraperitoneally injected to induce GSH depletion in rats. P-glycoprotein expression at the blood-brain barrier was examined by Western blotting and RT-PCR, and its function was assessed by measuring the brain-to-plasma concentration ratios (Kp values) of rhodamine 123 (Rh123). Evans Blue dye was used as a blood-brain barrier indicator for examining the extravasation from the blood to the brain. KEY FINDINGS: Four hours after treatment of rats with diethyl maleate, the brain GSH content significantly reduced. The mdr1a mRNA expression at the blood-brain barrier was upregulated, whereas no significant change in mdr1b mRNA expression was found. The P-glycoprotein level was significantly increased compared with control rats. At the same time, the Kp values of Rh123 suggested that function of P-glycoprotein was significantly enhanced at the blood-brain barrier in rats with GSH depletion induced by diethyl maleate. No significant difference of the Evans Blue dye concentration in the brain cortex was found between GSH depletion rats and control rats. Treatment of rats with N-acetylcysteine decreased P-glycoprotein upregulation induced by diethyl maleate. CONCLUSIONS: The oxidative stress induced by GSH depletion played a positive role in the regulation of function and expression of P-glycoprotein at the blood-brain barrier in rats.

Inhibition of P-glycoprotein function by procyanidine on blood-brain barrier.

The inhibitory effects of procyanidine, one of the components from the bark of Pinus massoniana Lamb, on the P-glycoprotein (P-gp) function of the blood-brain barrier (BBB) were studied using in vitro rat brain microvessel endothelial cells (RBMECs) and nude mice transplanted with human cerebroma. Quantitative accumulation and efflux of rhodamine 123 (Rh123), a P-gp substrate, were determined using a fluorescence spectrophotometer as a measure of P-gp function. Procyanidine markedly increased the accumulation of Rh123 by inhibiting its efflux in a dose-dependent manner. A 5-fold increase in cellular Rh123 was observed for procyanidine at 10 micromol/L. The verapamil-stimulated ATPase activity in plasma membrane vesicles from the RBMECs was estimated by measuring inorganic phosphate liberation. Procyanidine significantly inhibited the verapamil-induced P-gp ATPase activity by 78% when pretreated with 10 micromol/L in a concentration-dependent manner. The inhibition of P-gp by procyanidine was suggested to be at least partly due to its inhibition of P-gp ATPase. Procyanidine markedly improved the therapeutic effects of adriamycin (ADM) on nude mice transplanted with human cerebroma, compared with solitary treatment of ADM. The combination of 80 mg/kg procyanidine with 2 mg/kg ADM significantly elevated the days of survival with an increase in life span of 76%. The findings suggested that procyanidine was a potent inhibitor of P-gp on BBB and could improve the therapeutic effects on cerebral tumors of some drugs which are difficult to accumulate in the brain.

Relationship between insulin sensitivity index and cognitive function in diet-induced insulin resistant rats.

Clinical and animal studies have revealed significant cognitive impairment in type II diabetic subjects. However, whether there is a relationship between insulin resistance and cognitive function is poorly understood. In the present study, we used a high fat diet to induce insulin resistance (IR) in rats, insulin sensitivity index (ISI) (= FINS x FPG/22.5) to assess the extent of insulin resistance and the Morris Water Maze Task to judge cognitive function. The relationship between insulin sensitivity index and cognitive function was determined by analysing the correlation between ISI and the time rat spent in targeted quadrant, as well as between ISI and the times the rat swam across the very point where a platform was previously placed, using Pearson's method. Perfect negative correlation between ISI and cognitive function existed when ISI fell within a certain range, which indicates that insulin resistance is associated with cognitive function impairment in some cases where ISI might be an indicator.

Tooniliatone A sensitizes multidrug resistant cancer cells by decreasing Bcl-xL via activation of JNK MAPK signaling.

BACKGROUND: Multidrug resistance (MDR) refers to the phenotype of tumor cells that are resistant to various chemotherapeutic drugs with different structures and functions, which is clearly disadvantageous for patients. Finding a natural product that can effectively reverse the MDR of tumor cells is important for the treatment of patients. PURPOSE: To prove that tooniliatone A (TA), a novel typical limonoid, can effectively reverse the MDR of tumor cells and to explore its mechanism of action. METHODS: The MTT, CCK-8 and monoclonal formation assays, as well as flow cytometry, were used to evaluate the role of TA in reversing tumor multidrug resistance; then the mechanism of action for TA was explored by western blotting and real-time fluorescent quantitative PCR. RESULTS: TA significantly reversed the MDR of the K562/MDR and MCF-7/MDR cell lines. TA can inhibit the anti-apoptotic protein Bcl-xL to make cells sensitive to common chemotherapeutic drugs and activate the SAPK/JNK pathway to promote phosphorylation of JNK and its downstream cJun protein. Small interfering RNA-mediated knockdown of JNK and cJun could antagonize the MDR reversal effect of TA and the inhibition of Bcl-xL by TA. Therefore, we hypothesized that TA activates the JNK pathway to increase the transcription of the proapoptotic protein Bim, thereby inhibiting Bcl-xL and reversing MDR in tumor cells. CONCLUSION: Our study suggests that TA reverses tumor MDR by activating the SAPK/JNK pathway to inhibit the action of Bcl-xL. TA may be an effective tumor MDR reversal agent.

Vielanin P enhances the cytotoxicity of doxorubicin via the inhibition of PI3K/Nrf2-stimulated MRP1 expression in MCF-7 and K562 DOX-resistant cell lines.

BACKGROUND: Cancer cells that are resistant to structurally and mechanically unrelated anticancer drugs are said to have multidrug resistance (MDR). The overexpression of the ATP-binding cassette (ABC) transporter is one of the most important mechanisms of MDR. Vielanin P (VP), a dimeric guaiane from the leaves of Xylopia vielana, has the potential to reverse multidrug resistance. PURPOSE: To evaluate the meroterpenoid compound VP as a low cytotoxicity MDR regulator and the related mechanisms. METHODS: Cell viability was determined by CCK-8 and MTT assays. Apoptosis and the accumulation of doxorubicin (DOX) and 5(6)-carboxyfluorescein diacetate (CFDA) were determined by flow cytometry. We determined mRNA levels by quantitative real-time polymerase chain reaction (qRT-PCR). Protein levels were analyzed by Western blotting and immunofluorescence. RESULTS: In the MCF-7 and K562 DOX-resistant cell lines, VP treatment (10 µM or 20 µM) enhanced the activity of chemotherapeutic agents. We found that VP selectively inhibited MRP1 mRNA but not MDR1 mRNA. VP enhanced DOX-induced apoptosis and reduced colony formation in the presence of DOX in drug-resistant cells. Moreover, VP increased the accumulation of DOX and the MRP1-specific substrate CFDA. In addition, VP reversed MRP1 protein levels and the accumulation of DOX and CFDA in MRP1-overexpressing MCF-7 and K562 cells. Thus, the mechanism of MDR reversal by VP is MRP1-dependent. Furthermore, we found that the inhibitory effect of VP on MRP1 is PI3K/Nrf2-dependent. CONCLUSION: These results support the potential therapeutic value of VP as an MDR-reversal agent by inhibiting MRP1 via PI3K/Nrf2 signaling.

Reversal of multidrug resistance by icaritin in doxorubicin-resistant human osteosarcoma cells.

Multidrug resistance (MDR) is one of the major obstacles in cancer chemotherapy. Our previous study has shown that icariin could reverse MDR in MG-63 doxorubicin-resistant (MG-63/DOX) cells. It is reported that icariin is usually metabolized to icariside II and icaritin. Herein, we investigated the effects of icariin, icariside II, and icaritin (ICT) on reversing MDR in MG-63/DOX cells. Among these compounds, ICT exhibited strongest effect and showed no obvious cytotoxicity effect on both MG-63 and MG-63/DOX cells ranging from 1 to 10 µmol·L-1. Furthermore, ICT increased accumulation of rhodamine 123 and 6-carboxyfluorescein diacetate and enhanced DOX-induced apoptosis in MG-63/DOX cells in a dose-dependent manner. Further studies demonstrated that ICT decreased the mRNA and protein levels of multidrug resistance protein 1 (MDR1) and multidrug resistance-associated protein 1 (MRP1). We also verified that blockade of STAT3 phosphorylation was involved in the reversal effect of multidrug resistance in MG-63/DOX cells. Taken together, these results indicated that ICT may be a potential candidate in chemotherapy for osteosarcoma.

Physakengose G induces apoptosis via EGFR/mTOR signaling and inhibits autophagic flux in human osteosarcoma cells.

BACKGROUND: Physakengose G (PG) is a new compound first isolated from Physalis alkekengi var. franchetii, an anticarcinogenic traditional Chinese medicine. PG has shown promising anti-tumor effects, but its underlying mechanisms remain unknown. PURPOSE: To investigate the anti-cancer effects of PG on human osteosarcoma cells and the underlying mechanisms. METHODS: Cell viability was measured by MTT assay. Apoptosis rates, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) generation, and acidic vesicular organelles (AVOs) formation were determined by flow cytometry. Protein levels were analyzed by immunofluorescence and western blotting. RESULTS: PG inhibited cell proliferation and induced apoptosis in human osteosarcoma cells. PG treatment blocked EGFR phosphorylation and suppressed epidermal growth factor (EGF)-induced activation of downstream signaling molecules, such as AKT and mTOR. PG treatment resulted in lysosome dysfunction by altering lysosome acidification and LAMP1 levels, which led to autophagosome accumulation and autophagic flux inhibition. CONCLUSION: PG inhibits cell proliferation and EGFR/mTOR signaling in human osteosarcoma cells. Moreover, PG induces apoptosis through the mitochondrial pathway and impedes autophagic flux via lysosome dysfunction. Our findings indicate that PG has the potential to play a significant role in the treatment of osteosarcoma.