1.
CMR COLLEGE OF PHARMACY
Approved by ( AICTE and PCI),(Affiliated to JNTU Hyderabad)
Kandlakoya(V), Medchal(M),Hyderabad-501401
A seminar on:
Inducing apoptosis in cancer cell by natural compounds
and screening methods
SUBMITTED TO
JAWAHARLAL NEHRU TECHNOLOGICAL UNIVERSITY
HYDERABAD
In partial fulfillment of M. Pharmacy I Year, II semester
Presented By,
SYED DASTAGIR HUSSAIN
M. Pharm I Year,
Regn. No. 18T21S0108
Department of Pharmacology
Under the Guidance of:
Mrs. P. JYOTHI, M.Pharm ,
Assistant Professor ,
Department of Phamacology
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Contents
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1. Introduction of cancer
2. Apoptosis
3. Apoptosis in the treatment of cancer
4. Targets of apoptosis regulation
i. Death receptor ligands
ii. Bcl-2 pathway
iii. P53 pathway
5. Natural agent which induce Apoptosis in Cancer Cells
6. Drug which induce Apoptosis in Cancer Cells
7. Curcumine
8. Piperlongmine
9. Antitumor study in nude mice (in-vivo test)
10. Western blot (in-vitro test)
11. Conclusion

3.
Definition of a neoplasm or tumor is ‘a mass of tissue formed as a result of abnormal,
excessive, uncoordinated, autonomous and purposeless proliferation of cells even after
cessation of stimulus for growth which caused it’.[1]
Cancer
Cancer-related Genes and Cell Growth (Hallmarks of Cancer):
1. Excessive and autonomous growth: Growth-promoting oncogenes.
2. Refractoriness to growth inhibition: Growth suppressing anti-oncogenes.
3. Escaping cell death by avoiding apoptosis: Genes regulating apoptosis and cancer.
4. Avoiding cellular aging: Telomeres and telomerase in cancer.
5. Continued perfusion of cancer: Cancer angiogenesis.
6. Invasion and distant metastasis: Cancer dissemination.
7. DNA damage and repair system: Mutator genes and cancer.
8. Cancer progression and tumor heterogeneity: Clonal aggressiveness.
9. Cancer a sequential multistep molecular phenomenon: Multistep theory.
10. MicroRNAs in cancer: Onco miRs.[1]
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4.
CANCER-RELATED GENES AND CELL GROWTH
(HALLMARKS OF CANCER)[1]
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Fig.no:01 Cancer-related Genes and Cell Growth (Hallmarks of Cancer)

5.
APOPTOSIS
• The death of cells which occurs as a normal and controlled part of an organism's growth
or development.[2]
• It is a Greek word apo : from , ptosis : falling , apoptosis : falling off[2]
• For understanding of the mechanisms involved in the process of apoptosis in mammalian
cells transpired from the investigation of programmed cell death that occurs during the
development of the nematode Caenorhabditis elegans .[3]
• In this organism 1090 somatic cells are generated in the formation of the adult worm, of
which 131 of these cells undergo apoptosis or “programmed cell death.”
• These 131 cells die at particular points during the development process, which is
essentially invariant between worms, demonstrating the remarkable accuracy and control
in this system. [4]
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• Programmed cell death which involves the genetically determined elimination of cells.
• Apoptosis occurs normally during development and aging and as a homeostatic
mechanism to maintain cell populations in tissues.
• Apoptosis also occurs as a defense mechanism such as in immune reactions or when
cells are damaged by disease or noxious agents.
• Irradiation or drugs used for cancer chemotherapy results in DNA damage in some cells,
which can lead to apoptotic death through a p53-dependent pathway.
• Some hormones, such as corticosteroids, may lead to apoptotic death in some cells (e.g.,
thymocytes) although other cells are unaffected or even stimulated.
• Some cells express Fas or TNF receptors that can lead to apoptosis via ligand binding
and protein cross-linking.
• Other cells have a default death pathway that must be blocked by a survival factor such
as a hormone or growth factor.
IMPORTANCE :
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MECHANISMS OF APOPTOSIS[5]
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Fig.no:02. Types of apoptosis pathways

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Apoptosis in the treatment of cancer
• Most of the anticancer agents we use today were developed well before apoptosis .
• Most advanced novel therapies based on our apoptosis are still only at the clinical trial
stage.
• Established drugs, such as the steroid Dexamethasone it directly induce apoptosis of
cancer cells.
Targets of apoptosis regulation:
1. Death receptor ligands
2. Bcl-2 pathway
3. P53 pathway
4. Smac/Diablo mimetics
5. Herceptin, Gleevec and Iressa
6. Rituximab
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Death receptor pathway[6]
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Fig.no:03. Death receptor pathway

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BCL-2 family pathway[7]
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Fig.no:04. Bcl-2 pathway of apoptosis

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P-53 Pathway[8]
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Fig.no:05. P-53 pathway of apoptosis

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Natural agent which induce Apoptosis in Cancer Cells
1.Polyphenols
i. Resveratrol
ii. (-)-Epigallocatechin-3-gallate
iii. Curcumin
iv. Pterostilbene
3.Glycosides:
i. Picrocrocin
2.Alkaloids :
i. Piperlongmine
ii. Piperine
4.Flavonoids:
i. Apigenin
ii. Luteolin
iii. Quercetin
iv. chrysin
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Drug which induce Apoptosis in Cancer Cells
1. Actinomycin D
2. Tamoxifen
3. Vitamin E succinate
4. Arsenic trioxide
5. Nocodazole
6. Carboplatin
7. Anthraquinone derivative
• These are the drugs which induce apoptosis by indirect pathway
• Metabolites of drug effect on cancer cell as inducer of apoptosis
• These drugs are under trail for apoptosis activity
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Curcumine
• Curcumin is natural compound which is extract from turmeric
• Curcumin has received the GRAS status (generally recognized as safe) by the U.S FDA[9]
• It is polyphenols which induce apoptosis by regulating oncogenes and anti-oncogenes
• Induced apoptosis, inhibit NFκB activity, and downregulate expression of the antiapoptotic
Bcl-2, Bcl-xL, and XIAP proteins [10]
• Curcumin upregulates expression of P53, Bax, Bak, PUMA, Bim, NOXA and death receptors
DR4 and DR5, triggering activation of caspase-3, -9, and inducing polyadenosine
-5’diphosphate ribose polymerase (PARP) [11]
e.g. mantle cell lymphoma and multiple myeloma cell lines
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Mechanism of action of polyphenols on cancer cell
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Fig.no:06. Mechanism of action of curcumin on cancer cell

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Piperlongmine
• Piperlongumine (PL), a plant alkaloid, is known to selectively kill tumor cells while
sparing normal cells.
• The mechanism of PL-induced cancer cell death is an association of FOXO3A with the
pro-apoptotic protein BIM .[12]
• PL treatment induces FOXO3A dephosphorylation and nuclear translocation and
promotes its binding to the BIM gene promoter, resulting in the up-regulation of BIM in
the cancer cell lines.[13]
• siRNA mediated FOXO3A knockdown rescued the cells from PL-induced cell death. [14]
• In vivo, the PL treatment markedly inhibited xenograft tumor growth, and this inhibition
was accompanied by the activation of the FOXO3A-BIM axis.[15]
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Mechanism of action of piperlongam on cancer cell
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Fig.no:07. Mechanism of action of piperlongamine on cancer cell

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Antitumor study in nude mice (in-vivo test)[16]
1. Five-week-old female BALB/c nude mice (deficiency in T cell function allows
xenografts) (18~20 g)
2. MCF-7 cells were collected and injected subcutaneously (2 × 106 cells in 200 μL
normal saline) into the right flank of nude mice.
3. Six animals per group were used in each experiment.
4. When tumor mass reached 5-6 mm in diameter (7 days).
5. Mice were treated with sample by intra peritoneal (i.p.) injection once every other day
of induction.
6. Normal saline containing equal amount of DMSO was used as the vehicle control.
7. Tumor volumes were measured twice weekly using a vernier caliper, and the volume.
FORMULA:
𝝅
𝟔
× 𝒍𝒆𝒏𝒈𝒕𝒉 × 𝒘𝒊𝒅𝒕𝒉 𝒐𝒇 𝒕𝒖𝒎𝒐𝒓
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 At the end of experiment, the mice were sacrificed and the tumors were removed
and weighed for use in the protein expression studies.
1. Immunohistochemistry (IHC) staining.
2. Statistical analysis (ANOVA).
MCF-7 cells
5-6 mm in diameter of tumor
Inject test and standards
In alternative days
sacrificed
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Fig.no:08 Diagrammatic representation of anti-tumor activity test

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1. Cell lysates were prepared by extracting proteins with RIPA buffer (Sigma)
containing protease cocktail inhibitor (Roche, Basel, Switzerland).
2. Equal amounts of protein samples were subjected to SDS-PAGE and transferred
to a nitrocellulose membrane (Millipore, Billerica, MA, USA).
3. After blocking with 5% non-fat milk in Tris-buffered saline containing 0.1%
Tween-20 (TBST),
4. The membrane was incubated with primary antibodies diluted in blocking
solution (1:1,000) at 4˚C overnight.
5. After washing with TBST, the membrane was incubated for an additional 1 h with
the appropriate secondary antibodies conjugated to horseradish peroxidase at a
dilution of 1:5,000.
6. The protein bands were visualized using an enhanced chemilumine scence
detection system (GE Healthcare, Little Chalfont, Buckinghamshire, UK).
7. The density of the immuno reactive bands was analyzed using Image J software.
Western blot (in-vitro test)[17]
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Conclusion
 The goal of cancer therapy is to promote the death of cancer cells without causing too
much damage to normal cells.
 Apoptotic pathway has provided rationale targets for the development of new
anticancer therapeutics.
 Natural compounds as effective anti-cancer agents, both in the form of nutritional
supplements or functional foods, and as potential anticancer drugs.
 Mechanisms to prevent carcinogenesis include inhibition of pro-inflammatory
molecules generation, oxidative stress, DNA damage, and cancer cell proliferation,
and by increased apoptosis.
 We presenting polyphenol and alkaloids as natural product (curcumine and
piperlongamine) as apoptosis induer which has received the GRAS status (generally
recognized as safe) by the U.S FDA

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