Hispidulin induces ER stress-mediated apoptosis in human hepatocellular carcinoma cells in vitro and in vivo by activating AMPK signaling pathway.

Hispidulin (4',5,7-trihydroxy-6-methoxyflavone) is a phenolic flavonoid isolated from the medicinal plant S. involucrata, which exhibits anti-neoplastic activity against several types of cancer. However, the mechanism underlying its anti-cancer activity against hepatocellular carcinoma (HCC) has not been fully elucidated. In this study, we investigated whether and how hispidulin-induced apoptosis of human HCC cells in vitro and in vivo. We showed that hispidulin (10, 20 µmol/L) dose-dependently inhibited cell growth and promoted apoptosis through mitochondrial apoptosis pathway in human HCC SMMC7721 cells and Huh7 cells. More importantly, we revealed that its pro-apoptotic effects depended on endoplasmic reticulum stress (ERS) and unfolded protein response (UPR), as pretreatment with salubrinal, a selective ERS inhibitor, or shRNA targeting a UPR protein CHOP effectively abrogated hispidulin-induced cell apoptosis. Furthermore, we showed that hispidulin-induced apoptosis was mediated by activation of AMPK/mTOR signaling pathway as pretreatment with Compound C, an AMPK inhibitor, or AMPK-targeting siRNA reversed the pro-apoptotic effect of hispidulin. In HCC xenograft nude mice, administration of hispidulin (25, 50 mg/kg every day, ip, for 27 days) dose-dependently suppressed the tumor growth, accompanied by inducing ERS and apoptosis in tumor tissue. Taken together, our results demonstrate that hispidulin induces ERS-mediated apoptosis in HCC cells via activating the AMPK/mTOR pathway. This study provides new insights into the anti-tumor activity of hispidulin in HCC.

Hispidulin mediates apoptosis in human renal cell carcinoma by inducing ceramide accumulation.

Hispidulin, a polyphenolic flavonoid extracted from the traditional Chinese medicinal plant S involucrata, exhibits anti-tumor effects in a wide array of human cancer cells, mainly through growth inhibition, apoptosis induction and cell cycle arrest. However, its precise anticancer mechanisms remain unclear. In this study, we investigated the molecular mechanisms that contribute to hispidulin-induced apoptosis of human clear-cell renal cell carcinoma (ccRCC) lines Caki-2 and ACHN. Hispidulin (10, 20 µmol/L) decreased the viability of ccRCC cells in dose- and time-dependent manners without affecting that of normal tubular epithelial cells. Moreover, hispidulin treatment dose-dependently increased the levels of cleaved caspase-8 and caspase-9, but the inhibitors of caspase-8 and caspase-9 only partly abrogated hispidulin-induced apoptosis, suggesting that hispidulin triggered apoptosis via both extrinsic and intrinsic pathways. Moreover, hispidulin treatment significantly inhibited the activity of sphingosine kinase 1 (SphK1) and consequently promoted ceramide accumulation, thus leading to apoptosis of the cancer cells, whereas pretreatment with K6PC-5, an activator of SphK1, or overexpression of SphK1 significantly attenuated the anti-proliferative and pro-apoptotic effects of hispidulin. In addition, hispidulin treatment dose-dependently activated ROS/JNK signaling and led to cell apoptosis. We further demonstrated in Caki-2 xenograft nude mice that injection of hispidulin (20, 40 mg·kg-1·d-1, ip) dose-dependently suppressed tumor growth accompanied by decreased SphK1 activity and increased ceramide accumulation in tumor tissues. Our findings reveal a new explanation for the anti-tumor mechanisms of hispidulin, and suggest that SphK1 and ceramide may serve as potential therapeutic targets for the treatment of ccRCC.

Integration of MRI-Based Radiomics Features, Clinicopathological Characteristics, and Blood Parameters: A Nomogram Model for Predicting Clinical Outcome in Nasopharyngeal Carcinoma.

Purpose: This study aimed to develop a nomogram model based on multiparametric magnetic resonance imaging (MRI) radiomics features, clinicopathological characteristics, and blood parameters to predict the progression-free survival (PFS) of patients with nasopharyngeal carcinoma (NPC). Methods: A total of 462 patients with pathologically confirmed nonkeratinizing NPC treated at Sichuan Cancer Hospital were recruited from 2015 to 2019 and divided into training and validation cohorts at a ratio of 7:3. The least absolute shrinkage and selection operator (LASSO) algorithm was used for radiomics feature dimension reduction and screening in the training cohort. Rad-score, age, sex, smoking and drinking habits, Ki-67, monocytes, monocyte ratio, and mean corpuscular volume were incorporated into a multivariate Cox proportional risk regression model to build a multifactorial nomogram. The concordance index (C-index) and decision curve analysis (DCA) were applied to estimate its efficacy. Results: Nine significant features associated with PFS were selected by LASSO and used to calculate the rad-score of each patient. The rad-score was verified as an independent prognostic factor for PFS in NPC. The survival analysis showed that those with lower rad-scores had longer PFS in both cohorts (p < 0.05). Compared with the tumor-node-metastasis staging system, the multifactorial nomogram had higher C-indexes (training cohorts: 0.819 vs. 0.610; validation cohorts: 0.820 vs. 0.602). Moreover, the DCA curve showed that this model could better predict progression within 50% threshold probability. Conclusion: A nomogram that combined MRI-based radiomics with clinicopathological characteristics and blood parameters improved the ability to predict progression in patients with NPC.

Hispidulin inhibits hepatocellular carcinoma growth and metastasis through AMPK and ERK signaling mediated activation of PPARγ.

Hispidulin, a phenolic flavonoid, exerts potent cytotoxicity towards a variety of human cancers. However, the effects of hispidulin on hepatocellular carcinoma (HCC) and underlying molecular mechanisms of its action remain elusive. The present study investigated the effect of hispidulin on HCC in experimental models, including tumor cell lines and mouse tumor xenograft. Results demonstrated that hispidulin was cytotoxic and anti-proliferative to HCC cell lines (SMMC7721 and Bel7402). Hispidulin activated caspase-3 and triggered apoptosis in HCC cells. Moreover, hispidulin inhibited cell migration and invasion by inhibiting the expression of matrix metalloproteinases (MMP-2, MMP-9) and by inducing tissue inhibitor of metalloproteinase-3 (TIMP-3) expression. Hispidulin activated peroxisome proliferator-activated receptor γ (PPARγ) signaling which mainly contributed to its cytotoxicity in HCC cells. Remarkably, GW9662 (a PPARγ inhibitor) or PPARγ targeting siRNA significantly abrogated the anti-proliferative, pro-apoptotic, and anti-metastatic effects of hispidulin in HCC cells. Furthermore, hispidulin induced activation of PPARγ which was associated with increased phosphorylation of AMPK, ERK, JNK in HCC cells. Compound C (an AMPK inhibitor) or PD98059 (a MEK inhibitor) partly reversed the effects of hispidulin on PPARγ signaling in HCC cells. In contrast, no significant changes in PPARγ signaling were observed in HCC cells pretreated with SP600125 (a JNK inhibitor), while SP6000125 significantly inhibited the anti-cancer effects of hispidulin in HCC cells. Hispidulin administration effectively suppressed Bel7402 xenograft tumor growth and lung metastasis in vivo. Our findings indicate that PPARγ activation by hispidulin effectively suppressed HCC cell growth and metastasis both in vitro and in vivo.

Hispidulin suppresses tumor growth and metastasis in renal cell carcinoma by modulating ceramide-sphingosine 1-phosphate rheostat.

Sphingosine 1-phosphate (S1P) rheostat is considered as a key signal that determines cell fate. This study aimed to report that hispidulin, a polyphenolic flavonoid, exerted anti-growth and anti-metastasis effects against renal cell carcinoma (RCC) by modulating the balance of ceramide-S1P. In vitro studies showed that hispidulin could effectively inhibit cell proliferation, cell migration, cell invasion, and epithelial-mesenchymal transition, and promote cell apoptosis in Caki-2 and A498 cell lines. Moreover, it also increased the ceramide/S1P ratio. Consistent with the in vitro findings, the efficacy of hispidulin in vivo showed that it effectively suppressed tumor growth and lung metastasis. Furthermore, the results revealed that hispidulin significantly suppressed the activity of sphingosine kinase 1 (Sphk1) in RCC cells; however, no significant change was observed in the mRNA or protein expression of Sphk1. The overexpression of Sphk1 could significantly abrogate the anti-growth and anti-metastasis effects of hispidulin, whereas the siRNA-targeting Sphk1 or Sphk1 inhibitor was able to augment the anticancer effects of hispidulin against RCC. Moreover, hispidulin interfered with the phosphorylation and translocation of Sphk1, leading to inhibitory effects of Sphk1 activity. In summary, the findings suggested that hispidulin suppressed tumor growth and metastasis by inhibiting the Sphk1 activity and consequently modulating ceramide-S1P rheostat. It also presented a new explanation for the antitumor mechanisms of hispidulin against RCC.