Reversal of P-glycoprotein-mediated multidrug resistance is induced by mollugin in MCF-7/adriamycin cells
Tran TP, Kim HG, Choi JH, et al. Phytomedicine. 27 February 2013. doi:10.1016/j.phymed.2013.01.014
P-glycoprotein (P-gp), an important efflux transporter, is encoded by the MDR1 class of genes and is a central element of the multidrug resistance (MDR) phenomenon in cancer cells. In the present study, we investigated whether mollugin, purified from roots of Rubica cordifolia L., down-regulated MDR1 expression in MCF-7/adriamycin (MCF-7/adr) cells, a human breast multidrug-resistant cancer cell line.
Mollugin treatment significantly inhibited MDR1 expression by blocking MDR1 transcription. Mollugin treatment also significantly increased intracellular accumulation of the fluorescently-tagged P-gp substrate, rhodamine-123. The suppression of MDR1 promoter activity and protein expression was mediated through mollugin-induced activation of AMP-activated protein kinase (AMPK). Furthermore, mollugin inhibited MDR1 expression through the suppression of NF-κB and CREB activation.
Interestingly, mollugin also inhibited COX-2 expression. These results suggest that mollugin treatment enhanced suppression of P-gp expression by inhibiting the NF-κB signaling pathway and COX-2 expression, as well as attenuating CRE transcriptional activity through AMPK activation.
Mollugin inhibits proliferation and induces apoptosis by suppressing fatty acid synthase in HER2-overexpressing cancer cells
Do MT, Hwang YP, Kim HG, et al. Journal of Cellular Physiology. Volume 228, Issue 5, pages 1087–1097, May 2013. DOI: 10.1002/jcp.24258
Mollugin is a naphthohydroquine found in the roots of Rubia cordifolia, and has been reported to have a variety of biological activities, including anti-inflammatory and apoptotic effects. In the present study, we investigated the molecular mechanisms by which mollugin exerts anti-tumor effect in HER2-overexpressing cancer cells. Our results showed that mollugin exhibited potent inhibitory effects on cancer cell proliferation, especially in HER2-overexpressing SK-BR-3 human breast cancer cells and SK-OV-3 human ovarian cancer cells in a dose- and time-dependent manner without affecting immortalized normal mammary epithelial cell line MCF-10A. Furthermore, we found that a blockade of Akt/SREBP-1c signaling through mollugin treatment significantly reduced FAS expression and subsequently suppressed cell proliferation and induced apoptosis in HER2-overexpressing cancer cells. Mollugin treatment caused a dose-dependent inhibition of HER2 gene expression at the transcriptional level, potentially in part through suppression of NF-κB activation. The combination of mollugin with a MEK1/2 inhibitor may be required in order to achieve optimal efficacy in HER2-overexpressing cancers. These data provide evidence that mollugin inhibits proliferation and induces apoptosis in HER2-overexpressing cancer cells by blocking expression of the FAS gene through modulation of a HER2/Akt/SREBP-1c signaling pathway. Our findings suggest that mollugin is a novel modulator of the HER2 pathway in HER2-overexpressing cancer cells with a potential role in the treatment and prevention of human breast and ovarian cancer with HER2 overexpression.
Neuroprotective and anti-inflammatory effects of mollugin via up-regulation of heme oxygenase-1 in mouse hippocampal and microglial cells.
Jeong GS, Lee DS, Kim DC, et al. Eur J Pharmacol. 2011 Mar 11;654(3):226-34. doi: 10.1016/j.ejphar.2010.12.027. Epub 2011 Jan 13.
Mollugin, a bioactive phytochemical isolated from Rubia cordifolia L. (Rubiaceae), exhibits antimutagenic activity, antitumor activity, antiviral activity, and inhibitory activity in arachidonic acid- and collagen-induced platelet aggregation. In this study, we investigated the effects of mollugin as a neuroprotective agent in glutamate-induced neurotoxicity in the mouse hippocampal HT22 cell line and as an anti-inflammatory agent in lipopolysaccharide-induced microglial activation in BV2 cells. Mollugin showed potent neuroprotective effects against glutamate-induced neurotoxicity and reactive oxygen species generation in mouse hippocampal HT22 cells. In addition, the anti-inflammatory effects of mollugin were demonstrated by the suppression of pro-inflammatory mediators, including pro-inflammatory enzymes (inducible nitric oxide synthase and cyclooxygenase-2) and cytokines (tumor necrosis factor-α and interleukin-6). Furthermore, we found that the neuroprotective and anti-inflammatory effects of mollugin were linked to the up-regulation of the expression of heme oxygenase (HO)-1 and the activity of HO in HT22 and BV2 cells. In addition, the effects of mollugin resulted in the nuclear accumulation of nuclear factor-E2-related factor 2 (Nrf2) in HT22 and BV2 cells. Furthermore, mollugin also activated the p38 mitogen-activated protein kinase (MAPK) pathway both in HT22 and BV2 cells. These results suggest that mollugin may be a promising candidate for the treatment of neurodegenerative diseases related to neuroinflammation.
Shikonin Directly Targets Mitochondria and Causes Mitochondrial Dysfunction in Cancer Cells
Benjamin Wiench, Tolga Eichhorn, Malte Paulsen and Thomas Efferth. Evidence-Based Complementary and Alternative Medicine. Volume 2012 (2012), doi:10.1155/2012/726025
The naphthoquinone pigment shikonin is the most important pharmacologically active substance in the dried root of Lithospermum erythrorhizon. In traditional Chinese medicine root extracts of Lithospermum erythrorhizon have been used to treat macular eruption, measles, sore throat, carbuncles, and burns . The antitumor effect of shikonin was first evidenced by its activity against murine sarcoma-180 . A clinical trial using shikonin in 19 cases of late-stage lung cancer revealed that a shikonin-containing mixture was safe and effective for the treatment of late-stage cancer . The mechanism by which shikonin triggers its cytotoxic effect against malignant cells is controversial.
A very recent study showed that shikonin inhibits cancer cell glycolysis by targeting tumor pyruvate kinaseM2 . In this study, we show for the first time that the natural compound shikonin directly targets mitochondria causing mitochondrial dysfunction and ultimately apoptosis. We confirmed a number of recent findings regarding the cellular effects of shikonin, but our data suggests that most of them are downstream events. The primary effect of shikonin is the direct targeting of mitochondria, which causes a dose-dependent overproduction of ROS and an increase in intracellular calcium levels, leading to breakdown of the mitochondrial membrane potential and induction of the mitochondrial pathway of apoptosis. The increase in intracellular ROS levels and the mitochondrial injury cause other cellular effects such as oxidative DNA damage and inhibition of cancer cell migration.
We showed that shikonin has a strong cytotoxic effect on a wide variety of cancer cell lines, especially different types of leukemia and several known MDR cell lines. Microarray-based gene expression analysis of U937 leukemia cells suggested that the cytotoxicity of shikonin is based on the disruption of normal mitochondrial function, overproduction of ROS, inhibition of cytoskeleton formation, and finally induction of cell-cycle arrest and apoptosis. We were able to validate all of these effects using in vitro cell culture experiments exploiting the specific natural fluorescence of shikonin and thereby identify the possible primary cellular mechanism of shikonin’s cytotoxicity.
We support our claim with the finding that shikonin immediately accumulates in the mitochondria of cancer cells and disrupts mitochondrial function, as evidenced by the loss of mitochondrial membrane potential.
It was recently shown that shikonin induces ROS and apoptosis in cancer cells  and our results fully concur with this assertion. However, we would suggest that other previously described mechanisms of action for shikonin such as the induction of necroptosis , inhibition of topoisomerase II activity , downregulation of NFκB signaling , cell-cycle arrest through upregulation of p53 and downregulation of cyclin-dependent protein kinase 4 , inhibition of proteasome function , inhibition of tumor necrosis factor alpha , deregulation of calcium signaling, and microtubule disintegration are actually downstream effects mediated (in most cases) not by a direct interaction of shikonin with the suggested targets but rather by the direct generation of ROS, the subsequent dysregulation of mitochondria and induction of oxidative damage. A recent study showed that shikonin interferes with cancer cells’ energy generation by targeting tumor pyruvate kinase-M2 and thereby inhibiting glycolysis .
Our study confirms that shikonin treatment causes reduced energy production in cancer cells by affecting the mitochondrial membrane potential, but the observed effects of shikonin on ROS and mitochondrial function are not likely to be purely based on blocking glycolysis. If glycolysis is unable to serve as a source of acetyl-CoA for energy generation, cells can compensate by shift to other metabolic pathways such as fatty acid oxidation  or glutamine utilization .
Our data does not exclude the possibility that shikonin has an effect on pyruvate kinase-M2, but the direct targeting of mitochondria and the complete loss of the mitochondrial membrane potential as well as the rapid induction of ROS make the electron chain the more likely target of shikonin.Shikonin can be categorized as a mitocan , a class of compounds that act by interfering with energy-generating mitochondrial processes, which in turn leads to ROS accumulation, mitochondrial destabilization, and induction of apoptosis .
Shikonin itself is a naphthoquinone derivative, and various substituted naphthoquinones have been shown to be capable of redox cycling in isolated mitochondria . During this process, reductive enzymes, for example, mitochondrial NADH-ubiquinone oxidoreductase (complex 1), metabolize quinones to unstable semiquinones through one-electron reduction reactions .
When molecular oxygen is present, such semiquinones enter into a redox cycle leading to reformation of the original quinone, with the associated generation of reactive oxygen species. Ultimately, this cycle results in excessive ROS accumulation, depolarization of the mitochondrial membrane, and induction of apoptosis . Due to the quinone structure of shikonin and its accumulation in the mitochondria, we believe that the ROS induction caused by shikonin is also based on such a futile mitochondrial redox cycling. The elevated levels of ROS strain the mitochondria, leading to a breakdown of the mitochondrial membrane potential and finally to the release of proapoptotic compounds and thus the activation of caspases involved in the intrinsic pathway of apoptosis. The oxidative DNA damage detected is also a consequence of the elevated ROS production and could likely be the trigger of the observed cell-cycle arrest .Besides inducing ROS, some quinones have been shown to cause release of calcium from isolated mitochondria . This is consistent with the elevated levels of observed after shikonin treatment. We showed that shikonin, in contrast to ionomycin, caused a slow and continuous increase in intracellular calcium concentrations. This suggests that shikonin does not shuttle extracellular or intracellular stored calcium actively, but rather causes a calcium release from calcium stores or other organelles, for example, mitochondria, by an indirect mode such as via ROS signaling pathway . Nevertheless, elevated levels of and ROS together appreciably disturb normal calcium signaling .
Increased calcium levels promote the disassembly of microtubules by direct destabilization of growing microtubule ends , which is in accordance with our findings that shikonin inhibits cancer cell migration by the disruption of microtubule cytoskeleton dynamics. Indeed, shikonin treatment results in a complete inhibition of EB3 protein dynamics and a loss of distinct microtubule filaments, suggesting that the ATP shortage and deregulation of calcium levels are dually destructive. These findings motivate further investigations on the effect of shikonin in the treatment of highly invasive cancer types. Many established anticancer agents affect upstream signaling pathways that ultimately converge on mitochondria as regulators of cell death and survival . These signaling pathways are often deregulated in human cancers, and for this reason many MDR phenotypes are resistant to classical anticancer agents . Thus, compounds that directly target mitochondria can bypass deregulated upstream signaling events and thereby circumvent the resistance mechanisms of cancer cells .
However due to the basic mode of action it is likely that shikonin also has an effect on noncancer cells. Yet, shikonin bypasses resistances of known MDR cell types and this makes further research on better and more direct application methods an interesting project. Numerous animal studies showed that the therapeutic effects of shikonin apparently predominate the side effects [13, 53] and a clinical trial with shikonin showed that it can be utilized in therapy . Future studies should concentrate on the reduction of side effects by chemical derivatization or tissue targeted application.In summary, our results indicate that shikonin accumulates in the mitochondria of cancer cells, disrupts mitochondrial function, and finally causes apoptosis. As mitochondria generate the majority of the cellular ATP supply and also regulate the cell death machinery, they are promising targets for cancer therapy. Hence, shikonin may have potential for cancer
2. D. Hanahan and R. A. Weinberg, “Hallmarks of cancer: the next generation,” Cell, vol. 144, no. 5, pp. 646–674, 2011.
5. S. Fulda, L. Galluzzi, and G. Kroemer, “Targeting mitochondria for cancer therapy,” Nature Reviews Drug Discovery, vol. 9, no. 6, pp. 447–464, 2010.
6. P. S. Brookes, Y. Yoon, J. L. Robotham, M. W. Anders, and S. S. Sheu, “Calcium, ATP, and ROS: a mitochondrial love-hate triangle,” American Journal of Physiology—Cell Physiology, vol. 287, no. 4, pp. C817–C833, 2004
12. G. Guizzunti, E. A. Theodorakis, A. L. Yu, C. Zurzolo, and A. Batova, “Cluvenone induces apoptosis via a direct target in mitochondria: a possible mechanism to circumvent chemo-resistance?” Investigational New Drugs, vol. 30, no. 5, pp. 1841–1848, 2012.
13. X. Chen, L. Yang, J. J. Oppenheim, and O. M. Z. Howard, “Cellular pharmacology studies of shikonin derivatives,” Phytotherapy Research, vol. 16, no. 3, pp. 199–209, 2002.
14. U. Sankawa, Y. Ebizuka, T. Miyazaki, Y. Isomura, and H. Otsuka, “Antitumor activity of shikonin and its derivatives,” Chemical and Pharmaceutical Bulletin, vol. 25, no. 9, pp. 2392–2395, 1977.
15. X. P. Guo, X. Y. Zhang, and S. D. Zhang, “Clinical trial on the effects of shikonin mixture on later stage lung cancer,” Zhong Xi Yi Jie He Za Zhi, vol. 11, no. 10, pp. 598–580, 1991.
16. J. Chen, J. Xie, Z. Jiang, B. Wang, Y. Wang, and X. Hu, “Shikonin and its analogs inhibit cancer cell glycolysis by targeting tumor pyruvate kinase-M2,” Oncogene, vol. 30, pp. 4297–4306, 2011.
31. T. R. Henry and K. B. Wallace, “Differential mechanisms of induction of the mitochondrial permeability transition by quinones of varying chemical reactivities,” Toxicology and Applied Pharmacology, vol. 134, no. 2, pp. 195–203, 1995.
39. E. T. O'Brien, E. D. Salmon, and H. P. Erickson, “How calcium causes microtubule depolymerization,” Cell Motility and the Cytoskeleton, vol. 36, pp. 125–135, 1997.
41. N. Fujii, Y. Yamashita, Y. Arima, M. Nagashima, and H. Nakano, “Induction of topoisomerase II-mediated DNA cleavage by the plant naphthoquinones plumbagin and shikonin,” Antimicrobial Agents and Chemotherapy, vol. 36, no. 12, pp. 2589–2594, 1992. View at Scopus
42. Y. W. Cheng, C. Y. Chang, K. L. Lin, C. M. Hu, C. H. Lin, and J. J. Kang, “Shikonin derivatives inhibited LPS-induced NOS in RAW 264.7 cells via downregulation of MAPK/NF-κB signaling,” Journal of Ethnopharmacology, vol. 120, no. 2, pp. 264–271, 2008.
43. Z. Wu, L. Wu, L. Li, S. I. Tashiro, S. Onodera, and T. Ikejima, “p53-mediated cell cycle arrest and apoptosis Induced by Shikonin via a Caspase-9-Dependent Mechanism in Human Malignant Melanoma A375-S2 Cells,” Journal of Pharmacological Sciences, vol. 94, no. 2, pp. 166–176, 2004.
44. H. Yang, P. Zhou, H. Huang et al., “Shikonin exerts antitumor activity via proteasome inhibition and cell death induction in vitro and in vivo,” International Journal of Cancer, vol. 124, no. 10, pp. 2450–2459, 2009.
45. S. C. Chiu and N. S. Yang, “Inhibition of tumor necrosis factor-α through selective blockade of Pre-mRNA splicing by shikonin,” Molecular Pharmacology, vol. 71, no. 6, pp. 1640–1645, 2007.
46. M. Buzzai, D. E. Bauer, R. G. Jones et al., “The glucose dependence of Akt-transformed cells can be reversed by pharmacologic activation of fatty acid β-oxidation,” Oncogene, vol. 24, no. 26, pp. 4165–4173, 2005.
47. Y. Chendong, J. Sudderth, D. Tuyen, R. G. Bachoo, J. G. McDonald, and R. J. DeBerardinis, “Glioblastoma cells require glutamate dehydrogenase to survive impairments of glucose metabolism or Akt signaling,” Cancer Research, vol. 69, no. 20, pp. 7986–7993, 2009.
48. S. J. Ralph and J. Neuzil, “Mitochondria as targets for cancer therapy,” Molecular Nutrition & Food Research, vol. 53, no. 1, pp. 9–28, 2009.
49. T. Iyanagi and I. Yamazaki, “One-electron-transfer reactions in biochemical systems V. Difference in the mechanism of quinone reduction by the NADH dehydrogenase and the NAD(P)H dehydrogenase (DT-diaphorase),” Biochimica et Biophysica Acta, vol. 216, no. 2, pp. 282–294, 1970.
50. D. N. Criddle, S. Gillies, H. K. Baumgartner-Wilson et al., “Menadione-induced reactive oxygen species generation via redox cycling promotes apoptosis of murine pancreatic acinar cells,” Journal of Biological Chemistry, vol. 281, no. 52, pp. 40485–40492, 2006.
51. S. P. Jackson and J. Bartek, “The DNA-damage response in human biology and disease,” Nature, vol. 461, no. 7267, pp. 1071–1078, 2009.
51. G. Kroemer, L. Galluzzi, and C. Brenner, “Mitochondrial membrane permeabilization in cell death,” Physiological Reviews, vol. 87, no. 1, pp. 99–163, 2007.
52. G. Kroemer, L. Galluzzi, and C. Brenner, “Mitochondrial membrane permeabilization in cell death,” Physiological Reviews, vol. 87, no. 1, pp. 99–163, 2007
53. K. Gong and W. Li, “Shikonin, a Chinese plant-derived naphthoquinone, induces apoptosis in hepatocellular carcinoma cells through reactive oxygen species: a potential new treatment for hepatocellular carcinoma,” Free Radical Biology & Medicine, vol. 51, pp. 2259–2271, 2011