Patent Application: US-201514985419-A

Abstract:
the present invention discloses a method of treating cancer comprising administering an effective amount of an alkaloid , in which the alkaloid is liensinine , isoliensinine , dauricine , cepharanthine , hernandezine or thalidezine and isolated from the traditional chinese medicinal herbs . the use of the alkaloid in treating neurodegenerative disorder is also disclosed .

Description:
as used herein and in the claims , “ comprising ” means including the following elements but not excluding others . in this invention , a group of alkaloids including liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine are identified as novel inducers of autophagy . the chemical structures of these six alkaloids are demonstrated in fig1 a to 1 f respectively . studies conducted by inventors demonstrate that liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine can induce autophagy and autophagic cell death in a panel of cancer and apoptosis - resistant cells . on the other hand , these compounds are capable of promoting the degradation of mutant huntingtin with hdq55 or 74 cag repeats in pc12 cells . taken together , works by the inventors provide novel insights into the autophagic effect of selected alkaloids and their potential uses in anti - tumor or neuroprotective therapy in future . furthermore , in this invention , liensinine and isoliensinine are derived and isolated from seed embryos of nelumbo nucifera ; dauricine is derived and isolated from asiatic moonseed rhizome ; cepharanthine is derived and isolated from stephania cepharantha ; and hernandezine and thalidezine are derived and isolated from thalictrum podocarpum humb . the following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention . they should not be considered as limiting the scope of the invention , but merely as being illustrative and representative thereof . this example describes in vitro cell cytotoxicity of liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine in a panel of human cancer and normal cells . the test compounds of liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine were dissolved in dmso at a final concentration of 100 mmol / l and stored at − 20 ° c . cytotoxicity was assessed using the 3 -( 4 , 5 - dimethylthiazol - 2 - yl )- 2 , 5 - diphenyltetrazolium bromide assay as described previously [ 25 ]. 4000 - 8000 hela ( human cervical cancer ), mcf - 7 ( human breast cancer ), hepg2 ( human liver cancer ), hep3b ( human liver cancer ), h1299 ( human lung cancer ), a549 ( human lung cancer ), pc3 ( human prostate cancer ) and lo2 ( human normal liver ) cells were seeded on 96 - well plates per well . after overnight pre - incubation , the cells were exposed to different concentrations of liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine ( 0 . 039 - 100 μmol / l ) for 3 days . specifically , the following concentrations are used for all of the above alkaloids : 100 , 50 , 25 , 12 . 5 , 6 . 25 , 3 . 125 , 1 . 5625 , 0 . 78 , 0 . 39 , 0 . 195 , 0 . 079 , 0 . 039 μmol / l . subsequently , 10 μl of mtt reagents was added to each well and incubated at 37 ° c . for 4 hours followed by the addition of 100 μl solubilization buffer ( 10 % sds in 0 . 01 mol / l hcl ) and overnight incubation . absorbance at 585 nm was determined from each well on the following day . the percentage of cell viability was calculated using the following formula : cell viability (%)= cells number treated / cells number dmso control × 100 . data was obtained from three independent experiments . as shown in fig1 g , significant cell cytotoxicity was observed with mean ic 50 value ranging from 4 . 52 - 61 . 8 μm observed in a panel of human cancer cells treated with liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine for 72 hours as revealed by mtt assay . however , the test compounds of liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine indicated no or insignificant cytotoxic effect toward human normal liver lo2 cells . this example describes an in vitro study to demonstrate the autophagic effect of liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine . gfp - lc3 puncta formation was quantified as previously described [ 15 ]. in brief , gfp - lc3 transfected cells grown on coverslips in a 6 - well plate were treated with or without 20 μm of liensinine , 10 μm of isoliensinine , dauricine , cepharanthine , hernandezine or thalidezine for 4 hours , the cells were then fixed in 4 % paraformaldehyde for 20 minutes at room temperature and then rinsed with pbs . slides were mounted with fluorsave ™ mounting media ( calbiochem , san diego , calif .) and examined by fluorescence microscopy . the number of gfp - positive cells with gfp - lc3 puncta formation was examined under the nikon eclipse 80i microscope . representative images were captured with ccd digital camera spot rt3 ™ ( diagnostic instruments , inc ., melville , n . y .). to quantify for autophagy , the percentage of cells with punctate gfp - lc3 fluorescence was calculated by counting the number of the cells with punctate gfp - lc3 fluorescence in gfp - positive cells . a minimum of 150 cells from 3 randomly selected fields was scored . after treatments with liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine , cells were harvested and lysed in ripa buffer ( cell signaling technologies inc ., beverly , mass .). the cell lysates were then resolved by sds - page . after electrophoresis , the proteins from sds - page were transferred to nitrocellulose membrane which was then blocked with 5 % non - fat dried milk for 60 minutes . the membrane was then incubated with lc3 primary antibodies ( 1 : 1000 ) in tbst overnight at 4 ° c . after that , the membrane was further incubated with hrp - conjugated secondary antibodies for 60 minutes . finally , protein bands were visualized by using the ecl western blotting detection reagents ( invitrogen , paisley , scotland , uk ). 2 . 3 quantification of liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine - mediated autophagy in the presence of autophagic inhibitor . gfp - lc3 puncta formation was quantified as previously described [ 15 ]. in brief , hela cells expressing gfp - lc3 were treated with 20 μm of liensinine , or 10 μm of isoliensinine , dauricine , cepharanthine , hernandezine or thalidezine in the presence of autophagic inhibitor , 3 - methyl adenine ( 3 - ma , 5 mm ), for 4 hours . the cells were then fixed in 4 % paraformaldehyde for 20 minutes at room temperature and then rinsed with pbs . slides were mounted with fluorsave ™ mounting media ( calbiochem ) and examined by fluorescence microscopy . to quantify for autophagy , the percentage of cells with punctate gfp - lc3 fluorescence was calculated by counting the number of the cells with punctate gfp - lc3 fluorescence in gfp - positive cells . a minimum of 150 cells from 3 randomly selected fields were scored . as compared to dmso control treatment , liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine significantly induced the gfp - lc3 puncta formation in hela cancer cells as shown in fig2 a . western blot analysis showed that conversion of the autophagic marker lc3 - ii was also induced upon treatments of liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine as shown in fig2 b . in addition , liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine also increased the formation of gfp - lc3 puncta towards a panel of cancer and normal cells as revealed by fluorescent microscopy as shown in fig2 c . however , there was a significant reduction in the liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine - autophagy induced by gfp - lc3 puncta formation in hela cells in the presence of autophagic inhibitor ( 3 - ma ) as shown in fig3 , in which such findings were consistent with the gfp - lc3 puncta formation and lc3 conversion from lc3 - i to lc3 - ii as shown in fig2 a to 2 c . the data of this study suggested that liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine are the novel autophagy enhancers . although these compounds could induce autophagy in lo2 human normal liver cells , autophagy mediated by these compounds exhibits far less toxic in human normal liver cells as shown in fig1 g , suggesting that the cytotoxic effect mediated by liensinine , isoliensinine , dauricine , cepharanthine , hernandezine or thalidezine is tumor specific . study on dependency of the presence of autophagy - related gene 7 ( atg7 ) on autophagic effect this example describes an in vitro study to demonstrate that the autophagic effect of liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine is dependent on the presence of autophagy - related gene 7 ( atg7 ). 3 . 1 quantification of autophagy gfp - lc3 puncta in atg7 wild type and deficient mefs . gfp - lc3 puncta formation was quantified as previously described [ 15 ]. in brief , both atg7 wild - type ( atg7 - wt or atg7 +/+) and deficient ( atg7 −/−) mouse embryonic fibroblasts ( mefs ) were transfected with gfp - lc3 plasmid and then grown on coverslips in a 6 - well plate . the cells were then treated with 20 μm of liensinine , 10 μm of isoliensinine , 10 μm of dauricine , 10 μm of cepharanthine , hernandezine or 10 μm of thalidezine . for 24 h . the cells were then fixed in 4 % paraformaldehyde for 20 minutes at room temperature and then rinsed with pbs . slides were mounted with fluorsave ™ mounting media ( calbiochem , san diego , calif .) and examined by fluorescence microscopy . the number of gfp - positive cells with gfp - lc3 puncta formation was examined under the nikon eclipse 80i microscope . representative images were captured with ccd digital camera spot rt3 ™ ( diagnostic instruments , inc ., melville , n . y .). to quantify for autophagy , the percentage of cells with punctate gfp - lc3 fluorescence was calculated by counting the number of the cells with punctate gfp - lc3 fluorescence in gfp - positive cells . a minimum of 150 cells from 3 randomly selected fields were scored . liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine were found to induce gfp - lc3 puncta formation in wild type atg7 cells but not in atg7 - knockout ( atg7 - ko or atg7 −/−) mouse embryonic fibroblasts , as shown in fig4 a to 4 c . liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine work as the novel autophagy enhancers which depend on autophagy related gene , atg7 , for the induction of autophagy . in other words , the autophagy induced by the six aforementioned compounds was atg - 7 dependent . this example describes an in vitro study to demonstrate the mechanism and action of liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine during autophagy induction . hela cells treated with 20 μm of liensinine , 10 μm of isoliensinine , 10 μm of dauricine , 10 μm of cepharanthine , 10 μm of hernandezine and 10 μm of thalidezine were harvested and lysed in ripa buffer ( cell signaling ). the cell lysates were then resolved by sds - page . after electrophoresis , the proteins from sds - page were transferred to nitrocellulose membrane which was then blocked with 5 % non - fat dried milk for 60 minutes . the membrane was then incubated with p - p70s6k , p70s6k , p - ampk , ampk and actin primary antibodies ( 1 : 1000 ) in tbst overnight at 4 ° c . respectively . after that , the membrane was further incubated with hrp - conjugated secondary antibodies for 60 minutes . finally , protein bands were visualized by using the ecl western blotting detection reagents ( invitrogen ). 4 . 2 quantification of liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine - mediated autophagy in the presence of specific inhibitor . gfp - lc3 puncta formation was quantified as previously described [ 15 ]. in brief , hela cells expressing gfp - lc3 were treated with indicated concentrations of liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine in the presence of ampk inhibitor , compound c ( cc , 5 μm ), for 24 hours . the cells were then fixed in 4 % paraformaldehyde for 20 minutes at room temperature and then rinsed with pbs . slides were mounted with fluorsave ™ mounting media ( calbiochem ) and examined by fluorescence microscopy . to quantify for autophagy , the percentage of cells with punctate gfp - lc3 fluorescence was calculated by counting the number of the cells with punctate gfp - lc3 fluorescence in gfp - positive cells . a minimum of 150 cells from 3 randomly selected fields were scored . liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine were found to activate the phosphorylation of ampk as compared to dmso control treatment as shown in fig5 a and this activation was also accompanied by a concomitant reduction in its downstream p70s6k phosphorylation . in order to confirm whether the ampk signaling is involved in autophagy induced by liensinine , isoliensinine , dauricine , cepharanthine , hernandezine or thalidezine , specific ampk inhibitor , compound c , was used in the study . results showed that there was a significant reduction in the gfp - lc3 puncta formation induced by liensinine , isoliensinine , dauricine , cepharanthine , hernandezine or thalidezine in hela cells treated with the presence of ampk inhibitor ( compound c ), as shown in fig5 b , suggesting that the ampk signaling is required for autophagy induction by these alkaloid compounds . liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine are shown to induce autophagy via modulation of ampk - mtor signaling pathway . this example describes an in vitro study to demonstrate that liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine induce autophagic cell death in cells . cell viability was measured using an annexin v staining kit ( bd biosciences , san jose , calif ., usa ). briefly , atg7 wild - type ( atg7 +/+ or atg7 - wt ) and atg7 deficient ( atg7 −/− or atg7 - ko ) mouse embryonic fibroblasts ( mefs ) were treated with the selected alkaloids for 24 h . cells were then harvested and analysed by multiparametric flow cytometry using fitc - annexin v and propidium iodide staining ( bd biosciences , san jose , calif ., usa ) according to the manufacturer &# 39 ; s instructions . flow cytometry was then carried out using a facscalibur flow cytometer ( bd biosciences , san jose , calif ., usa ). data acquisition and analysis was performed with cellquest ( bd biosciences , san jose , calif ., usa ). data were obtained from three independent experiments . among the six tested compounds , as shown in fig6 a to 6 c , all alkaloids are found to exhibit less cytotoxic effect to autophagy deficient cells ( atg7 −/− or atg7 - ko ). the results suggest that liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine induce cell death or cell cytotoxicity via autophagy induction . this example describes an in vitro study to demonstrate that isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine potently induce cell cytotoxicity in apoptosis - resistant cells . the test compounds of isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine were dissolved in dmso at a final concentration of 100 mmol / l and stored at − 20 ° c . cytotoxicity was assessed using the 3 -( 4 , 5 - dimethylthiazol - 2 - yl )- 2 , 5 - diphenyltetrazolium bromide assay as previously described [ 25 ]. 2500 of caspase wild - type ( caspase wt ), caspase - 3 deficient ( caspase 3ko ), caspase - 7 deficient ( caspase 7k0 ), caspase - 3 /- 7 deficient ( caspase 3 / 7 dko ), caspase - 8 deficient ( caspase 8k0 ), bax - bak wild - type ( bak - bak wt ) and bax - bak double knock out ( bak - bak dko ) mouse embryonic fibroblasts ( mefs ) were seeded on 96 - well plates per well . after overnight pre - incubation , the cells were exposed to different concentrations of isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine ( 0 . 039 - 100 μmol / l ) for 3 days . specifically , the following concentrations are used for all of the above alkaloids : 100 , 50 , 25 , 12 . 5 , 6 . 25 , 3 . 125 , 1 . 5625 , 0 . 78 , 0 . 39 , 0 . 195 , 0 . 079 , 0 . 039 μmol / l . subsequently , 10 μl of mtt reagents was added to each well and incubated at 37 ° c . for 4 hours , followed by the addition of 100 μl solubilization buffer ( 10 % sds in 0 . 01 mol / l hcl ) and overnight incubation . absorbance at 585 nm was determined from each well on the following day . the percentage of cell viability was calculated using the following formula : cell viability (%)= cells number treated / cells number dmso control × 100 . data was obtained from three independent experiments . isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine are found to exhibit similar cytotoxic effect on both wild - type and apoptosis - resistant cells , i . e . caspase - 3 /- 7 /- 8 as compared to the caspase wild - type mefs as shown in fig7 a to 7 e ). in addition , from fig7 a to 7 e , similar cytotoxicity is also shown in bax - bak dko apoptosis - resistant cells as compared to bax - bak wild - type mefs , indicating that these alkaloid compounds are able to induce cell death in apoptosis - resistant cells . these findings suggest that isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine are capable to induce cell cytotoxicity in apoptosis - resistant cancer cells . this example describes an in vitro study to demonstrate the clearance of mutant huntingtin hdq55 / 74 by liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine . for cell viability assay measured by crystal violet staining , pc - 12 cells were incubated in 35 mm disc followed by the addition of liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine at 5 - 10 μm for 24 h . the cells were then incubated with crystal violet for 10 minutes followed by a ddh 2 o wash . images of the stained cells were captured by ccd digital camera spot rt3 ™ under the nikon eclipse 80i microscope with 4 × magnification . cell viability was quantified by dissolving stained cells in 10 % acetic acid ( 200 μl / well ). the colorimetric reading of the solute mixture was then determined by spectrophotometer at od 560 nm . the percentage of cell viability was calculated using the following formula : cell viability (%)= cells number treated / cells number dmso control × 100 . data was obtained from three independent experiments . pc 12 cells were transfected transiently with egfp - hdq55 / 74 plasmids for 24 h using lipofectamine plus ltx reagent ( invitrogen ) according to the manufacturer &# 39 ; s protocol . the transfected cells were then treated with liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine for 24 h . the removal of mutant huntingtin , ( hdq55 & amp ; hdq74 ) was then quantitated by immunoblotting with antibody against egfp or by immunocytochemistry under fluorescence microscopy . as shown in fig8 a , liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine exhibit no toxicity in pc 12 at 5 - 10 μm . in addition , 5 - 10 μm of liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine enhanced the clearance of overexpressed egfp - tagged mutant huntingtin ( hdq55 , hdq74 ) with 55 and 74 cag repeats as measured by immunoblotting against egfp antibody as shown in fig8 b . concomitantly , fluorescence imaging as illustrated in fig8 c further revealed that the six aforesaid compounds significantly reduced the formed mutant huntingtin aggregate ( hdq55 ) in pc 12 cells . liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine may work as a novel neuroprotective agent through accelerating the clearance of mutant huntingtin . the present invention relates to the identification of a group of novel autophagy enhancers , namely , liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine , which are isolated from chinese medicinal herbs , nelumbo nucifera ( liensinine and isoliensinine ), asiatic moonseed rhizome ( dauricine ), stephania cepharantha ( cepharanthine ), thalictrum hernandezii ( hernandezine ) and thalictrum podocarpum humb ( thalidezine ) respectively . the invention also covers the anti - cancer effect of the above alkaloid compounds through induction of autophagic cell death in a panel of cancer cells and apoptosis - resistant cells . in addition , the invention further covers the neuroprotective effect of the above compounds on neuronal cells via enhancing the clearance of mutant huntingtin . in one embodiment of the present invention , liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine exhibit significant cytotoxic effect towards a panel of cancer cells , but not in human normal liver lo2 cells . in the further embodiment of the present invention , liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine exhibit specific cytotoxic effect toward human cancer cells . in one embodiment of the present invention , liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine are the novel autophagy enhancers and never be reported before . in the further embodiment of the present invention , liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine are capable to induce autophagy in a panel of cancer and normal cells , and animals . in one embodiment of the present invention , autophagy induced liensinine , isoliensinine , dauricine , cepharanthine , hernandezine or thalidezine is dependent on autophagy - related gene 7 ( atg7 )). in the further embodiment of the present invention , liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine are capable to induce autophagy in atg7 dependent manner in one embodiment of the present invention , liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine induce autophagy via activation of amp - activated protein kinase ( ampk ) and inhibition of mammalian target of rapamycin ( mtor ) signaling . in the further embodiment of the present invention , liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine are capable to induce autophagy via modulation of ampk - mtor signaling pathway . in one embodiment of the present invention , liensinine , isoliensinine , dauricine and thalidezine are found to exhibit less cytotoxicity in autophagy deficient cells ( atg7 −/−), indicating that liensinine , isoliensinine , dauricine and thalidezine are able to induce autophagic cell death in wild - type atg7 cells . in the further embodiment of the present invention , liensinine , isoliensinine , dauricine and thalidezine are capable to induce autophagic cell death mechanism in atg7 containing cancer cells . in another embodiment of the present invention , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine exhibit significant cytotoxic effect towards a panel of apoptosis - resistant cells . in the further embodiment of the present invention , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine exhibit potent cytotoxic effect towards apoptosis - resistant cancer cells . in another embodiment of the present invention , liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine enhance the clearance of mutant huntingtin hdq55 / 74 in pc12 cells . in the further embodiment of the present invention , liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine are capable to enhance the clearance of mutant huntingtin . the preferred embodiment of the present invention , liensinine , isoliensinine , dauricine , cepharanthine , hernandezine and thalidezine could be developed as novel anti - cancer and neuroprotective agents for patients with cancers or neurodegenerative diseases . in another embodiment , the neurodegenerative diseases can be selected from alzheimer &# 39 ; s disease , parkinson &# 39 ; s disease , huntington &# 39 ; s disease , amyotrophic lateral sclerosis , ataxia telangiectasia , spinocerebellar atrophy and multiple sclerosis . the exemplary embodiments of the present invention are thus fully described . although the description referred to particular embodiments , it will be clear to one skilled in the art that the present invention may be practiced with variation of these specific details . hence this invention should not be construed as limited to the embodiments set forth herein . 1 . levine , b ., and g . kroemer . 2008 . autophagy in the pathogenesis of disease . cell 132 : 27 - 42 . 2 . pallauf , k ., and g . rimbach . autophagy , polyphenols and healthy ageing . ageing res rev 12 : 237 - 252 . 3 . rubinsztein , d . c ., j . e . gestwicki , l . o . murphy , and d . j . klionsky . 2007 . potential therapeutic applications of autophagy . nat rev drug discov 6 : 304 - 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