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Betulinic Acid



RESEARCH


Betulinic acid (BetA) belongs to the lupane class of pentacyclic triterpenoid that was first isolated from the stem bark of Zizyphus mauritiana in 1995 [43] and has been found in many plants viz. Tryphyllum peltaum, Ancistrocladus heyneaus, Zizyphus joazeiro, Diospyoros leucomelas, Tetracera boliviana, and Syzygium formosanum[43]. The anticancer activity of BetA has been demonstrated in both in vitro and in vivo tumor models as well as in human clinical trials. Pisha et al. identified BetA as a melanoma specific cytotoxic agent that especially induced apoptosis of melanoma cells in cultures and inhibited the growth of human melanoma tumors implanted subcutaneously in athymic mice without any associated systemic toxicity [43]. Subsequent studies with BetA showed selective cytotoxicity on a variety of tumor cell lines but not on normal cells [44] and induced apoptosis in neuroblastoma and glioblastoma cells through the activation of the mitochondrial pathway [45] and [46]. Interestingly, a report by Tan et al. showed that apoptosis induced by BetA was mediated by activation of MAPK kinase pathway without involvement of activated caspases [47]. Activated NF-κB and STAT3 is frequently observed in tumor cells proliferation, invasion, metastasis and angiogenesis. Takada and Aggarwal, investigated the effect of BetA on constitutive and inducible NF-κB activation pathway induced by a variety of stimulants such as TNF-α, PMA, cigarette smoke, okadaic acid, IL-1, and H2O2 in H1299 cells (lung adenocarcinoma), HCT 116 and Caco-2 cells (colon carcinoma) [48]. They found that BetA suppressed IkBα kinase activation and subsequent phosphorylation of IkBα and p65 translocation to the nucleus. This correlated with the down-regulation of various proliferative and anti-apoptotic and metastatic gene products including COX-2, cyclin D1, Bcl-2, Bcl-xL and MMPs. Chintharlapalli et al. showed that BetA inhibited prostate cancer growth through inhibition of the transcription factors, specificity protein1 (Sp1), Sp3, and Sp4 which regulate VEGF and survivin expression [49]. Furthemore, BetA has been shown to inhibit constitutive activation of STAT3, Src kinase, JAK1 and JAK2 and induce the expression of the protein tyrosine phosphatase, SHP-1. Silencing of the SHP-1 gene abolished the ability of BetA to inhibit STAT3 activation and also rescued tumor cells from BetA-induced cell death. BetA also significantly downregulated the expression of STAT3 regulated gene products such as, Bcl-xL, Bcl-2, cyclin D1 and survivin [50]. 23-hydroxy betulinic acid, isolated from Pulsatilla chinensis induced apoptosis of HL-60 cells by downregulating Bcl-2 and telomerase activity and of colorectal cancer cells by modulating mitochondrial membrane potential [51]. In a cell-free system, BetA has been demonstrated to directly cause mitochondrial outer membrane permeabilization and cytochrome-c release in a Bcl-2 or Bcl-xL-dependent manner, yet independently of caspases [52], [53] and [54]. BetA has also been reported to induce apoptosis in a p53-independent fashion, including chemotherapy-refractory cases [44], [45], [54], [55], [56], [57] and [58] indicating that this tritepene can also overcome chemoresistance, which is frequently encountered during cancer therapy suggesting tremendous potential for development as a therapeutic agent. Recent evidence indicates that the anticancer activity of BetA can be markedly increased when it is used in combination with conventional chemotherapy, ionizing radiation or cytokine TRAIL [57], [59] and [60]. Furthermore BetA analogs have displayed significant anti-inflammatory activities in various animal models [61]. Fukuda et al. showed that oral administration of BetA prevented ultravioletB-induced photocarcinogenesis [62]. Similarly, NVX-207, a synthetic derivative of BetA, was shown to have potent anti-cancer activity. NVX-207 at 3.5 μM showed anti-tumor activity against various human and canine cell lines, and induced apoptosis by activation of caspase-3. Global gene expression profiling demonstrated that NVX-207 upregulated genes coding for lipid metabolism and cholesterol transport. A phase I/II study in dogs suffering from naturally occurring cancer receiving topical treatment of NVX-207 (10 mg/ml) showed excellent clinical responses including a complete remission in all animals receiving treatment. NVX-207 is also well tolerated and has significant anti-cancer activity both in vitro and in vivo[63].
References:
    [43] E. Pisha, H. Chai, I.S. Lee, T.E. Chagwedera, N.R. Farnsworth, G.A. Cordell, C.W. Beecher, H.H. Fong, A.D. Kinghorn, D.M. Brown et al. Discovery of betulinic acid as a selective inhibitor of human melanoma that functions by induction of apoptosis. Nat. Med., 1 (1995), pp. 1046–1051

   [44] V. Zuco, R. Supino, S.C. Righetti, L. Cleris, E. Marchesi, C. Gambacorti-Passerini, F. Formelli. Selective cytotoxicity of betulinic acid on tumor cell lines, but not on normal cells. Cancer Lett., 175 (2002), pp. 17–25

   [45] S. Fulda, C. Friesen, M. Los, C. Scaffidi, W. Mier, M. Benedict, G. Nunez, P.H. Krammer, M.E. Peter, K.M. Debatin. Betulinic acid triggers CD95 (APO-1/Fas)- and p53-independent apoptosis via activation of caspases in neuroectodermal tumors. Cancer Res., 57 (1997), pp. 4956–4964

   [46] I. Jeremias, H.H. Steiner, A. Benner, K.M. Debatin, C. Herold-Mende. Cell death induction by betulinic acid, ceramide and TRAIL in primary glioblastoma multiforme cells. Acta Neurochir. (Wien), 146 (2004), pp. 721–729

   [47] Y. Tan, R. Yu, J.M. Pezzuto. Betulinic acid-induced programmed cell death in human melanoma cells involves mitogen-activated protein kinase activation. Clin. Cancer Res., 9 (2003), pp. 2866–2875

   [48] Y. Takada, B.B. Aggarwal. Betulinic acid suppresses carcinogen-induced NF-kappa B activation through inhibition of I kappa B alpha kinase and p65 phosphorylation: abrogation of cyclooxygenase-2 and matrix metalloprotease-9. J. Immunol., 171 (2003), pp. 3278–3286

   [49] S. Chintharlapalli, S. Papineni, S.K. Ramaiah, S. Safe. Betulinic acid inhibits prostate cancer growth through inhibition of specificity protein transcription factors. Cancer Res., 67 (2007), pp. 2816–2823

   [50] M.K. Pandey, B. Sung, B.B. Aggarwal. Betulinic acid suppresses STAT3 activation pathway through induction of protein tyrosine phosphatase SHP-1 in human multiple myeloma cells. Int. J. Cancer. J. Int. du Cancer, 127 (2010), pp. 282–292

   [51] Z.N. Ji, W.C. Ye, G.G. Liu, W.L. Hsiao. 23-Hydroxybetulinic acid-mediated apoptosis is accompanied by decreases in bcl-2 expression and telomerase activity in HL-60 Cells. Life Sci., 72 (2002), pp. 1–9

   [52] S. Fulda, C. Scaffidi, S.A. Susin, P.H. Krammer, G. Kroemer, M.E. Peter, K.M. Debatin. Activation of mitochondria and release of mitochondrial apoptogenic factors by betulinic acid. J. Biol. Chem., 273 (1998), pp. 33942–33948

   [53] S. Fulda, G. Kroemer. Targeting mitochondrial apoptosis by betulinic acid in human cancers. Drug Discov. Today, 14 (2009), pp. 885–890

   [54] S. Fulda, K.M. Debatin. Betulinic acid induces apoptosis through a direct effect on mitochondria in neuroectodermal tumors. Med. Pediatr. Oncol., 35 (2000), pp. 616–618

   [55] G.I. Salti, J.V. Kichina, T.K. Das Gupta, S. Uddin, L. Bratescu, J.M. Pezzuto, R.G. Mehta, A.I. Constantinou. Betulinic acid reduces ultraviolet-C-induced DNA breakage in congenital melanocytic naeval cells: evidence for a potential role as a chemopreventive agent. Melanoma Res., 11 (2001), pp. 99–104

   [56] R.D. Meng, W.S. El-Deiry. P53-independent upregulation of KILLER/DR5 TRAIL receptor expression by glucocorticoids and interferon-gamma.  Exp. Cell Res., 262 (2001), pp. 154–169

   [57] E. Selzer, E. Pimentel, V. Wacheck, W. Schlegel, H. Pehamberger, B. Jansen, R. Kodym. Effects of betulinic acid alone and in combination with irradiation in human melanoma cells. J. Invest. Dermatol., 114 (2000), pp. 935–940

   [58] W. Wick, C. Grimmel, B. Wagenknecht, J. Dichgans, M. Weller. Betulinic acid-induced apoptosis in glioma cells: a sequential requirement for new protein synthesis, formation of reactive oxygen species, and caspase processing. J. Pharmacol. Exp. Ther., 289 (1999), pp. 1306–1312

   [59] S. Fulda, I. Jeremias, K.M. Debatin. Cooperation of betulinic acid and TRAIL to induce apoptosis in tumor cells. Oncogene, 23 (2004), pp. 7611–7620

   [60] S. Fulda, K.M. Debatin. Sensitization for anticancer drug-induced apoptosis by betulinic acid. Neoplasia, 7 (2005), pp. 162–170

   [61] T. Honda, K.T. Liby, X. Su, C. Sundararajan, Y. Honda, N. Suh, R. Risingsong, C.R. Williams, D.B. Royce, M.B. Sporn, G.W. Gribble Design, synthesis, and anti-inflammatory activity both in vitro and in vivo of new betulinic acid analogues having an enone functionality in ring A. Bioorg. Med. Chem. Lett., 16 (2006), pp. 6306–6309

   [62] Y. Fukuda, K. Sakai, S. Matsunaga, H. Tokuda, R. Tanaka. Cancer chemopreventive effect of orally administrated lupane-type triterpenoid on ultraviolet light B induced photocarcinogenesis of hairless mouse. Cancer Lett., 240 (2006), pp. 94–101

   [63] M. Willmann, V. Wacheck, J. Buckley, K. Nagy, J. Thalhammer, R. Paschke, T. Triche, B. Jansen, E. Selzer. Characterization of NVX-207, a novel betulinic acid-derived anti-cancer compound. Eur. J. Clin. Invest., 39 (2009), pp. 384–394

Anti-leukaemic activity of Dillenia indica L. fruit extract and quantification of betulinic acid by HPLC.
The methanolic extract of Dillenia indica L. fruits showed significant anti-leukaemic activity in human leukaemic cell lines U937, HL60 and K562. This finding led to fractionation of the methanolic extract, on the basis of polarity, in which the ethyl acetate fraction showed the highest anti-leukemic activity. A major compound, betulinic acid, was isolated from the ethyl acetate fraction by silica gel column chromatography and was identified and characterized. Betulinic acid could explain the anti-leukemic activity of the methanolic extract and the ethyl acetate fraction. Hence the quantitative estimation of betulinic acid was approached in methanolic extract and fractions using HPLC.
Kumar D, Mallick S, Vedasiromoni JR, Pal BC. Phytomedicine. 2010 May;17(6):431-5. Epub 2009 Aug 12.

Chemical and biological investigations of Dillenia indica Linn.
A total of four compounds namely, lupeol (1), betulinaldehyde (2), betulinic acid (3) and stigmasterol (4) were isolated from the stem extract of Dillenia indica Linn. The structures of the isolated compounds (1-4) were established by extensive spectroscopic studies. The crude methanolic extracts and its n-hexane, carbon tetrachloride, dichloromethane and chloroform soluble partitionates demonstrated weak antimicrobial activity against a wide range of gram-positive and gram-negative bacteria and fungi. The extractives revealed significant cytotoxic activity when tested by brine shrimp lethality bioassay. In addition, the extractives exhibited significant free radical scavenging activity when compared with the standard drug ascorbic acid.
Most PN, Rahman MS, Islam MS, Rashid MA. Bangladesh Journal of Pharmacology, Vol 4, No 2 (2009)

Betulinic acid-induced apoptosis in leukemia cells
Betulinic acid (BA), a natural component isolated from Birch trees, effectively induces apoptosis in neuroectodermal and epithelial tumor cells and exerts little toxicity in animal trials. Here, we show that BA-induced marked apoptosis in 65% of primary pediatric acute leukemia cells and all leukemia cell lines tested. When compared for in vitro efficiency with conventionally used cytotoxic drugs, BA was more potent than nine out of 10 standard therapeutics and especially efficient in tumor relapse. No crossresistances were found between BA and any cytotoxic drug. Intracellular apoptosis signaling in leukemia tumor cells paralleled the pathway found in neuroectodermal cells involving caspases, but not death receptors. In isolated mitochondria, BA induced release of both cytochrome c and Smac. Taken together, BA potently induces apoptosis in leukemia cells and should be further evaluated as a future drug to treat leukemia.
EhrhardtH, Fulda S Führer M, Debatin KM & Jeremias I. Leukemia (2004) 18, 1406–1412. doi:10.1038/sj.leu.2403406

Betulinic Acid Triggers CD95 (APO-1/Fas)- and p53-independent Apoptosis via Activation of Caspases in Neuroectodermal Tumors.
Betulinic acid (BA), a melanoma-specific cytotoxic agent, induced apoptosis in neuroectodermal tumors, such as neuroblastoma, medulloblastoma, and Ewing's sarcoma, representing the most common solid tumors of childhood. BA triggered an apoptosis pathway different from the one previously identified for standard chemotherapeutic drugs. BA-induced apoptosis was independent of CD95-ligand/receptor interaction and accumulation of wild-type p53 protein, but it critically depended on activation of caspases (interleukin 1ß-converting enzyme/Ced-3-like proteases). FLICE/MACH (caspase-8), considered to be an upstream protease in the caspase cascade, and the downstream caspase CPP32/YAMA/Apopain (caspase-3) were activated, resulting in cleavage of the prototype substrate of caspases PARP. The broad-spectrum peptide inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, which blocked cleavage of FLICE and PARP, also completely abrogated BA-triggered apoptosis. Cleavage of caspases was preceded by disturbance of mitochondrial membrane potential and by generation of reactive oxygen species. Overexpression of Bcl-2 and Bcl-xl conferred resistance to BA at the level of mitochondrial dysfunction, protease activation, and nuclear fragmentation. This suggested that mitochondrial alterations were involved in BA-induced activation of caspases. Furthermore, Bax and Bcl-xs, two death-promoting proteins of the Bcl-2 family, were up-regulated following BA treatment. Most importantly, neuroblastoma cells resistant to CD95- and doxorubicin-mediated apoptosis were sensitive to treatment with BA, suggesting that BA may bypass some forms of drug resistance. Because BA exhibited significant antitumor activity on patients' derived neuroblastoma cells ex vivo, BA may be a promising new agent for the treatment of neuroectodermal tumors in vivo.
Fulda S, Friesen C, Los M et al. Cancer Research 57, 4956-4964, November 1, 1997

Selective cytotoxicity of betulinic acid on tumor cell lines, but not on normal cells. Volume 175, Issue 1, Pages 17-25 (10 January 2002)
Valentina Zuco, Sabina C Righetti, Loredana Cleris, Edoardo Marchesi, Carlo Gambacorti-Passerini, Franca Formelli

Betulinic acid is a triterpene with selective cytotoxicity against melanoma, neuroectodermal and malignant brain tumor cell lines. In this study the betulinic acid activity was evaluated, in comparison with doxorubicin, on different human neoplastic and non-neoplastic cell lines and on proliferating normal lymphocytes. Growth inhibition was evident in all the neoplastic cell lines independently on p53 status and histotype. Antiproliferative activity of betulinic acid was related to a cytotoxic effect on two p53 wild-type and on one p53 mutant cell lines and to a cytostatic effect on one p53 mutant melanoma clone. At the same concentrations, normal cells were unaffected indicating a selective effect of this agent. A cytotoxic activity of doxorubicin was evident on all the tested systems. In vivo experiments, performed on one of these cell lines, confirmed the antineoplastic activity of this drug. These data support further preclinical studies of betulinic acid not confined to melanoma and neuroectodermal tumours independently of p53 status.


Chemistry, biological activity, and chemotherapeutic potential of betulinic acid for the prevention and treatment of cancer and HIV infection. Medicinal Research Reviews. Volume 24 Issue 1, Pages 90 - 114 22 Oct 2003. DOI:10.1002/med.10053
Robert H. Cichewicz & Samir A. Kouzi

3-Hydroxy-lup-20(29)-en-28-oic acid (betulinic acid) is a pentacyclic lupane-type triterpene that is widely distributed throughout the plant kingdom. A variety of biological activities have been ascribed to betulinic acid including anti-inflammatory and in vitro antimalarial effects. However, betulinic acid is most highly regarded for its anti-HIV-1 activity and specific cytotoxicity against a variety of tumor cell lines. Interest in developing even more potent anti-HIV agents based on betulinic acid has led to the discovery of a host of highly active derivatives exhibiting greater potencies and better therapeutic indices than some current clinical anti-HIV agents. While its mechanism of action has not been fully determined, it has been shown that some betulinic acid analogs disrupt viral fusion to the cell in a post-binding step through interaction with the viral glycoprotein gp41 whereas others disrupt assembly and budding of the HIV-1 virus. With regard to its anticancer properties, betulinic acid was previously reported to exhibit selective cytotoxicity against several melanoma-derived cell lines. However, more recent work has demonstrated that betulinic acid is cytotoxic against other non-melanoma (neuroectodermal and malignant brain tumor) human tumor varieties. Betulinic acid appears to function by means of inducing apoptosis in cells irrespective of their p53 status. Because of its selective cytotoxicity against tumor cells and favorable therapeutic index, even at doses up to 500 mg/kg body weight, betulinic acid is a very promising new chemotherapeutic agent for the treatment of HIV infection and cancer. Med Res Rev, 24, No. 1, 90-114, 2004


Broad in vitro efficacy of plant-derived betulinic acid against cell lines derived from the most prevalent human cancer typeVolume 251, Issue 1, Pages 132-145 (18 June 2007)
Jan H. Kesslera1, Franziska B. Mullauera1, Guido M. de Roob, Jan Paul Medemaa

Betulinic acid (BA) is a widely available plant-derived triterpene with reported activity against cancer cells of neuroectodermal origin and leukaemias. Treatment with BA was shown to protect mice against transplanted human melanoma and led to tumor regression. In contrast, cells from healthy tissues were resistant to BA and toxic side-effects in animals were absent. These findings have raised interest in the chemotherapeutical anti-cancer potential of BA.
A comprehensive assessment of the efficacy of BA against the clinically most important cancer types is currently lacking. Therefore, we tested the in vitro sensitivity of broad cell line panels derived from lung, colorectal, breast, prostate and cervical cancer, which are the prevalent cancer types characterized with highest mortalities in woman and men. Multiple assays were used in order to allow a reliable assessment of anti-cancer efficacy of BA. After 48h of treatment with BA, cell viability as assessed with MTT and cell death as measured with propidium iodide exclusion showed clear differences in sensitivity between cell lines. However, in all cell lines tested colony formation was completely halted at remarkably equal BA concentrations that are likely attainable in vivo. Our results substantiate the possible application of BA as a chemotherapeutic agent for the most prevalent human cancer types


Betulinic acid: A new cytotoxic agent against malignant brain-tumour cells. International Journal of Cancer. 1999 Volume 82 Issue 3, Pages 435 - 441
Simone Fulda 1, Irmela Jeremias 2, Hans H. Steiner 3, Torsten Pietsch 4, Klaus-Michael Debatin

Malignant brain tumours are the most common solid tumors in children. The overall prognosis for this group of patients is still poor, emphasizing the importance of more effective therapies. Betulinic acid (Bet A) has been described as a novel cytotoxic compound active against melanoma and neuroblastoma cells. Here we report that Bet A was active against medulloblastoma and glioblastoma cell lines. In addition, Bet A exerted cytotoxic activity against primary tumor cells cultured from patients in 4 of 4 medulloblastoma-tumor samples tested and in 20 of 24 glioblastoma-tumor samples. Since a small percentage of primary-glioblastoma-tumor cells (4/24) did not respond to Bet-A treatment, resistance to Bet A might occur. Induction of apoptosis by Bet A involved mitochondrial perturbations, since inhibition of the mitochondrial permeability transition by the mitochondrion-specific inhibitor bongkrekic acid (BA) reduced Bet-A-induced apoptosis. In addition, mitochondria undergoing Bet-A-induced permeability transition triggered DNA fragmentation in isolated nuclei. Cytochrome c was released from mitochondria of Bet-A-treated cells, and might be involved in activation of caspases. Following treatment with Bet A, caspase-8, caspase-3 and PARP were proteolytically processed. Inhibition of caspase cleavage by the broad-range caspase inhibitor zVAD.fmk strongly reduced Bet-A-induced apoptosis, indicating that apoptosis was mediated by activation of caspases. Since Bet A did not exhibit cytotoxicity against murine neuronal cells in vitro, these findings suggest that Bet A may be a promising new agent for the treatment of medulloblastoma and glioblastoma cells that clearly warrants further pre-clinical and clinical evaluation. Int. J. Cancer 82:435-441, 1999.


Betulinic acid derivatives as HIV-1 antivirals. Trends in Molecular Medicine. Volume 11, Issue 1, January 2005, Pages 31-6. doi:10.1016/j.molmed.2004.11.001   
Christopher Aikena, E-mail The Corresponding Author and Chin Ho Chen

Betulinic acid (BA) derivatives are low molecular weight organic compounds synthesized from a plant-derived natural product. Several BA derivatives are potent and highly selective inhibitors of HIV-1. Depending on the specific side-chain modification, these compounds function by inhibiting HIV fusion or, as recently demonstrated, by interfering with a specific step in HIV-1 maturation. BA derivatives have potential as novel HIV-1 therapies, and additional studies of their mechanisms of action are likely to further define the novel targets of these compounds and elucidate the basic biology of HIV-1 fusion and maturation. In this review, recent studies of the novel mechanisms of action of this interesting class of antiviral compounds are discussed.


Effects of Betulinic Acid Alone and in Combination with Irradiation in Human Melanoma Cells. Journal of Investigative Dermatology (2000) 114, 935–940; doi:10.1046/j.1523-1747.2000.00972.x
Edgar Selzer*, Emilio Pimentel*, Volker Wacheck†, Werner Schlegel*, Hubert Pehamberger‡,§, Burkhard Jansen†,‡ and Reinhard Kodym
Recently, betulinic acid was identified as a highly selective inhibitor of human melanoma growth and was reported to induce apoptosis in these cells. We have investigated the growth-inhibitory properties of this compound alone and in combination with ionizing radiation in a panel of established human melanoma cell lines as well as in normal human melanocytes. Betulinic acid strongly and consistently suppressed the growth and colony-forming ability of all human melanoma cell lines investigated. In combination with ionizing radiation the effect of betulinic acid on growth inhibition was additive in colony-forming assays. Betulinic acid also induced apoptosis in human melanoma cells as demonstrated by Annexin V binding and by the emergence of cells with apoptotic morphology. The growth-inhibitory action of betulinic acid was more pronounced in human melanoma cell lines than in normal human melanocytes. Notably, despite the induction of apoptosis, analysis of the expression of Bcl-2 family members in betulinic-acid-treated cells revealed that expression of the anti-apoptotic protein Mcl-1 was induced. Furthermore, the antiproliferative action of betulinic acid seemed to be independent of the p53 status. The properties of betulinic acid make it an interesting candidate, not only as a single agent but also in combination with radiotherapy. We conclude that the strictly additive mode of growth inhibition in combination with irradiation suggests that the two treatment modalities may function by inducing different cell death pathways or by affecting different target cell populations.


Phytomedicine. 2010 May;17(6):431-5. Epub 2009 Aug 12.
Anti-leukaemic activity of Dillenia indica L. fruit extract and quantification of betulinic acid by HPLC.
Kumar D, Mallick S, Vedasiromoni JR , Pal BC.
National Institute of Pharmaceutical Education and Research, 4, Raja S.C.
Mullick Road, Jadavpur, Kolkata 700032, India.
Abstract
The methanolic extract of Dillenia indica L. fruits showed significant anti-leukaemic activity in human leukaemic cell lines U937, HL60 and K562. This finding led to fractionation of the methanolic extract, on the basis of polarity, in which the ethyl acetate fraction showed the highest anti-leukemic activity. A major compound, betulinic acid, was isolated from the ethyl acetate fraction by silica gel column chromatography and was identified and characterized. Betulinic acid could explain the anti-leukemic activity of the methanolic extract and the ethyl acetate fraction. Hence the quantitative estimation of betulinic acid was approached in methanolic extract and fractions using HPLC.

Chemical and biological investigations of Dillenia indica Linn.
Bangladesh Journal of Pharmacology, Vol 4, No 2 (2009)
Most. Nazma Parvin, Mohammad S. Rahman, Mohammad S. Islam, Mohammad A. Rashid
Abstract
A total of four compounds namely, lupeol (1), betulinaldehyde (2), betulinic acid (3) and stigmasterol (4) were isolated from the stem extract of Dillenia indica Linn. The structures of the isolated compounds (1-4) were established by extensive spectroscopic studies. The crude methanolic extracts and its n-hexane, carbon tetrachloride, dichloromethane and chloroform soluble partitionates demonstrated weak antimicrobial activity against a wide range of gram-positive and gram-negative bacteria and fungi. The extractives revealed significant cytotoxic activity when tested by brine shrimp lethality bioassay. In addition, the extractives exhibited significant free radical scavenging activity when compared with the standard drug ascorbic acid.
Leukemia (2004) 18, 1406–1412. doi:10.1038/sj.leu.2403406 Published online 17 June 2004


Betulinic acid-induced apoptosis in leukemia cells
H Ehrhardt1, S Fulda2, M Führer1, K M Debatin2 and I Jeremias1
Abstract
Betulinic acid (BA), a natural component isolated from Birch trees, effectively induces apoptosis in neuroectodermal and epithelial tumor cells and exerts little toxicity in animal trials. Here, we show that BA-induced marked apoptosis in 65% of primary pediatric acute leukemia cells and all leukemia cell lines tested. When compared for in vitro efficiency with conventionally used cytotoxic drugs, BA was more potent than nine out of 10 standard therapeutics and especially efficient in tumor relapse. No crossresistances were found between BA and any cytotoxic drug. Intracellular apoptosis signaling in leukemia tumor cells paralleled the pathway found in neuroectodermal cells involving caspases, but not death receptors. In isolated mitochondria, BA induced release of both cytochrome c and Smac. Taken together, BA potently induces apoptosis in leukemia cells and should be further evaluated as a future drug to treat leukemia.

Betulinic Acid Triggers CD95 (APO-1/Fas)- and p53-independent Apoptosis via Activation of Caspases in Neuroectodermal Tumors.
[Cancer Research 57, 4956-4964, November 1, 1997]
Simone Fulda, Claudia Friesen, Marek Los, Carsten Scaffidi, Walter Mier, Mary Benedict, Gabriel Nuñez, Peter H. Krammer, Marcus E. Peter and Klaus-Michael Debatin2
Betulinic acid (BA), a melanoma-specific cytotoxic agent, induced apoptosis in neuroectodermal tumors, such as neuroblastoma, medulloblastoma, and Ewing's sarcoma, representing the most common solid tumors of childhood. BA triggered an apoptosis pathway different from the one previously identified for standard chemotherapeutic drugs. BA-induced apoptosis was independent of CD95-ligand/receptor interaction and accumulation of wild-type p53 protein, but it critically depended on activation of caspases (interleukin 1ß-converting enzyme/Ced-3-like proteases). FLICE/MACH (caspase-8), considered to be an upstream protease in the caspase cascade, and the downstream caspase CPP32/YAMA/Apopain (caspase-3) were activated, resulting in cleavage of the prototype substrate of caspases PARP. The broad-spectrum peptide inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, which blocked cleavage of FLICE and PARP, also completely abrogated BA-triggered apoptosis. Cleavage of caspases was preceded by disturbance of mitochondrial membrane potential and by generation of reactive oxygen species. Overexpression of Bcl-2 and Bcl-xl conferred resistance to BA at the level of mitochondrial dysfunction, protease activation, and nuclear fragmentation. This suggested that mitochondrial alterations were involved in BA-induced activation of caspases. Furthermore, Bax and Bcl-xs, two death-promoting proteins of the Bcl-2 family, were up-regulated following BA treatment. Most importantly, neuroblastoma cells resistant to CD95- and doxorubicin-mediated apoptosis were sensitive to treatment with BA, suggesting that BA may bypass some forms of drug resistance. Because BA exhibited significant antitumor activity on patients' derived neuroblastoma cells ex vivo, BA may be a promising new agent for the treatment of neuroectodermal tumors in vivo.


Betulinic acid: a promising anticancer candidate.
Betulinic acid is a naturally occurring pentacyclic triterpenoid which has demonstrated selective cytotoxicity against a number of specific tumor types, a variety of infectious agents such as HIV, malaria and bacteria, and the inflammatory process in general. Biological activity was first demonstrated in melanoma cell lines and was confirmed in mice bearing human melanoma xenografts. These in vivo studies also established a favorable safety margin for betulinic acid, as systemic side effects were not observed at any dose. Recently, considerable in vitro evidence has demonstrated that betulinic acid is effective against small- and non-small-cell lung, ovarian, cervical, and head and neck carcinomas. Published data suggest that betulinic acid induces apoptosis in sensitive cells in a p53- and CD95-independent fashion. While the precise molecular target and mechanism of action remain elusive and are the focus of a number of ongoing research programs, accumulated experimental evidence indicates that betulinic acid functions through a mitochondrial-mediated pathway. Supplemental reports suggest that the generation of reactive oxygen species, inhibition of topoisomerase I, activation of the MAP kinase cascade, inhibition of angiogenesis, and modulation of pro-growth transcriptional activators and aminopeptidase N activity may play a role in betulinic acid-induced apoptosis. These potential mechanisms of action may enable betulinic acid to be effective in cells resistant to other chemotherapeutic agents. Arguments supporting the role of this agent in the treatment of cancers and other infectious conditions will be reviewed.
Eiznhamer DA, Xu ZQ. IDrugs. 2004 Apr;7(4):359-73.


Betulinic acid as new activator of NF-kappaB: molecular mechanisms and implications for cancer therapy
Recent evidence demonstrates that the anticancer activity of betulinic acid (BetA) can be markedly increased by combination protocols, for example with chemotherapy, ionizing radiation or TRAIL. Since nuclear factor-kappaB (NF-kappaB), a key regulator of stress-induced transcriptional activation, has been implicated in mediating apoptosis resistance, we investigated the role of NF-kappaB in BetA-induced apoptosis. Here, we provide for the first time evidence that BetA activates NF-kappaB in a variety of tumor cell lines. NF-kappaB DNA-binding complexes induced by BetA consisted of p50 and p65 subunits. Nuclear translocation of p65 was also confirmed by immunofluorescence microscopy. BetA-induced NF-kappaB activation involved increased IKK activity and phosphorylation of IkappaB-alpha at serine 32/36 followed by degradation of IkappaB-alpha. Reporter assays revealed that NF-kappaB activated by BetA is transcriptionally active. Interestingly, inhibition of BetA-induced NF-kappaB activation by different chemical inhibitors (proteasome inhibitor, antioxidant, IKK inhibitor) attenuated BetA-induced apoptosis. Importantly, specific NF-kappaB inhibition by transient or stable expression of IkappaB-alpha super-repressor inhibited BetA-induced apoptosis in SH-EP neuroblastoma cells, while transient expression of IkappaB-alpha super-repressor had no influence on BetA-induced apoptosis in two other cell lines. Thus, our findings that activation of NF-kappaB by BetA promotes BetA-induced apoptosis in a cell type-specific fashion indicate that NF-kappaB inhibitors in combination with BetA would have no therapeutic benefit or could even be contraproductive in certain tumors, which has important implications for the design of BetA-based combination protocols.
Kasperczyk H, La Ferla-Brühl K, Westhoff MA, et a. Oncogene (2005) 24, 6945–6956. doi:10.1038/sj.onc.1208842; published
 
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