1. EXPLOITING LACTAPTIN, A BREAST MILK
PEPTIDE IN CANCER THERAPY
P r e s e n t e d b y :
OLADOKUN Oladiran Boniface
SCP12/13/H/0032
BIOCHEMISTRY DEPARTMENT,
O B A FEMI A WO LO WO U N I VERSI TY,
I LE - I FE, O SU N STATE, NI G ERI A

2.  Cancer: more than 100 diseases characterized by excessive, uncontrolled growth of
abnormal cells, which invade and destroy other tissues.
 Resulting from compound dysregulations in cell replication
 Cancer represents a serious scourge to humans;
 Affects one in every three persons born in developed countries and is a major cause
of sickness and death throughout the world.
 Ranked the second leading cause of death, exceeded only by heart disease
 The National Cancer Institute of the United States (NCI) put up an estimate that more
than 10 million Americans are living with cancer (Peterson, 2009).
 Able to emerge in any part of the body as long as they are made of replicating cells; breast,
prostate, lung, colorectal, bladder, skin etc.
 Efforts to put an end to this scourge have not yielded a total victory because of the
different elusive attributes of cancers, and diverse biochemical and molecular activities
deployed in cancer evoking derangements that are peculiar to the emergence and
perpetuation of cancer cells.
Liver C22 3,385 204 420 97 4,106
Lung C33-C34 28,166 1,928 4,178 912 35,184
Death as a result of cancer in England, Wales, Scotland
Cancer Research, UK 2013

3. Ability to turn deaf ear to anti-growth signals
Invade other parts of the body
Recruit moreblood vesselsfor oxygen andnutrients
transport
Manipulate the cell’s clock that
saysa cellhas divided enough
E V A D E A P O P T O S I S
 Ability tomanipulate thetight leashed growthsignaling control system resulting in
endless flow of growth signals
Lactaptin is a pro-apoptotic protein
and also an antagonist of pro-cancer autophagy

4. KEY METABOLIC PROCESSES OF INTEREST
A P O P T O S I S
A u t o p h a g y

5. Apoptosis can be defined as a controlled demolition process that ensures the safe dismantling
of cellular structures and removal of the resulting debris such that collateral damage to
surrounding tissue is minimized.
To achieve this, the agents of destruction must be strictly controlled to ensure that they are
not deployed under the wrong circumstances, or in the wrong order. As with any complex and
potentially hazardous task, cellular demolition is best left in the hands of specialized molecules
that will coordinate the process and also be coordinated to minimize the likelihood of
disorderliness.
Rather than dying chaotically, cells undergo apoptosis in response to intrinsic or extrinsic
signals which rang when cells are infected with viruses, bacteria or when mutations are
detected and there is need to prevent such mutation from spreading, also when free radicals
are let loose causing cellular stress or when a person is exposed to radiations
A P O P TO S I S

6. T H E M A J O R E V E N T S I N A P O P T O S I S
http//:www.howstuffworks.com

7. C A S P A S E S : MEDIATORS OF APOPTOSIS
Since the events of apoptosis must be well controlled and tightly regulated, there is the need
for a well ordered COMPLEX CASCADE involving various molecules.
Caspases are known modulators of apoptosis.
Caspases, a family evolutionary conserved cysteinyl aspartate-specific proteases, are central
mediators of apoptotic and inflammatory pathways. Caspases are synthesized as zymogens.
Studies have established the activation of some caspases in response to lactaptin

8. INTRINSIC PATHWAY EXTRINSIC PATHWAY
F A D D
F A S
Silenced
!
DISC

9. APOPTOSIS AS A CONCERTED EFFORT
DEATH RECEPTORS; Fas, TNFR etc.
DEATH ADAPTORS; FADD etc.
CASPASES:
Initiators: 2, 8, 9, 10, 11, 12 etc. recipient of pro-apoptotic
flowing signals.
Effectors: 3, 6,7; get activated by initiator caspases
and in turn activate apoptosis.
BAX AND BAK: Bax antagonizes anti-apoptotic bcl-2 protein
Upregulated by p53
Upon apoptotic signals-becomes organelle-membrane associated
Possible route of action:
Disruption of voltage channel in the mitochondrion
Formation of pores (aided by BAK) leading to outer mitochodrion
permeabilization. These events are triggers for caspases.

10. KEY METABOLIC PROCESSES OF INTEREST
A p o p t o s i s
A U T O P H A G Y
Fluorescently labeled autophagosomes in liver cells of starved mice
http://en.wikipedia.org/wiki/File:Autophagy_diagram_PLoS_Biology.jpg

11. T H E P A R A D O X O F A U T O P H A G Y
 Autophagy is a cellular degradation process employed by cells for clearance of damaged or
more than necessary proteins and organelles
 A noble mission when deployed to degrade its content (in salvaging homeostasis and steady
energy in extremities) and its toxic waste
 A very important pathway for the survival of cells. Under physiological conditions autophagy
serves as one of the cellular mechanisms maintaining homeostasis by the degradation of cellular
components, misfolded, aggregated proteins or damaged organelles (Levine and Klionsky, 2004).
 Studies have shown over the last five years that a cell without autophagy cannot survive
(Mortensen et al., 2010; Phadwal et al., 2012).
 Implicated in the etiology of diseases e.g. Parkinson's; characterized by the accumulation of
protein aggregates in neuronal cells
 Also, as we age, wrinkles, hearing loss and other features of ageing may be due to fall in
autophagy levels and accumulation of toxic wastes in the cells (Mortensen et al., 2010; Phadwal
et al., 2012).
 PARADOXICALLY, this noble mission of autophagy has been implicated in cancers’ ability to
thrive.

12. Ryan and Noboru, 2010
Targeted cytoplasmic constituents are isolated from the rest of the cell within the autophagosome,
which are then fused with lysosomes and degraded or recycled.
Atg 7
Atg 1
Acidic lysosomal hydrolases
Beclin 1 Atg 9
Atg 10
LC3

13. A high autophagy will increase tumor cells survival
even in the over-choked –ever growing tumor
community (Karantza et al., 2007).
Dysregulated autophagy may result in accelerated
cell death (Lum et al., 2005).
Blockage of autophagy can thus facilitate
apoptosis (Carew et al., 2007).
Targeting cell death by apoptosis is thus one of the most exploited strategy in the design of anticancer
drugs (Meng et al., 2006). This is what makes Lactaptin worthy of interest.

14. A S O L U T I O N F R O M T H E
B R E A S T M I L K
Breast milk contains many bioactive proteins
Some become active following partial proteolysis (Brück et al., 2014).
Lactaptin is a proteolytic fragment of the residues 57–134 of human milk kappa-casein

15. PREVIOUS CLEAVAGE OF MILK’S
k-CASEIN
Casein exist predominately in the form of micelles and are recovered from skim milk by
using a mineral acid at low pH or Lactobacillus that converts milk sugar to lactic acid and
promotes precipitation of casein.
Casein micelle in milk forms a unique bio-colloid from calcium, phosphate, and proteins.
Four main types of proteins are involved: alpha(s1)-casein (38 %), alpha(s2)-casein
(10%), beta-casein (36 %) and kappa-casein (13%).
Isolated human kappa casein has been fragmented using chymosin by previous
researchers

16. OTHER MODIFICATIONS OF k-
CASEIN
--46 – 55
--54 – 59
--56 – 62
--79 – 82
ALL HAVE IMMUNO-MODULATORY FUNCTIONS
Mercier et al., 1973
Bergstroem et al., 1992)
Plowman et al., 1990
Human breast milk kappa casein (Mammary gland specific): 182 amino acid chain
length, further modified by O-glycosylation with GalNAc at different positions to yield
different variants.
Isolation from milk: Iso-electric precipitation of whole casein + gel chromatography on
sephadex G-200 in the presence of SDS + removal of SDS+ FPLC
(Dev et al., 1993).

17. L A C T - A P T I N
A potential success to the decades of the study of proteolysis on milk protein and recently one of the focus of cancer research .
The proteolytic fragment of human kappa casein with amino acid residues; 57 – 134
LACTAPTIN is known to cause the death of cultured cancer cells (Nekipelaya et al. 2008; Semenov et al., 2010)
Semenov et al., 2010 used this peptide in effecting apoptosis in human breast adenocarcinoma cell line (MCF-
7 cell lines).
Non-malignant human mesenchymal stem cells are completely resistant to lactaptin action(Semenov et al.,
2010)
 Induced phosphatidylserine externalization
 Not only Induces apoptosis of tumor cells in vitro but also inhibits tumor growth and metastasis of various
tumours (Koval et al., 2012)
Template for RL2: a recombinant analogue

18. M E C H A N I S M S O F A C T I O N O F L A C T A P T I N
CASPASE 3 & 7 activation
Phosphatidylserine normally resides on the
surface of the internal plasma membrane
bilayer
The interior PS get exposed in pathological
condions, during platelet activation, as part of
cellular recognition by macrophages
PS is externalized prior to apoptosis
Initiator caspases (including caspase-2, -8, -9, -
10, -11, and -12) are closely coupled to pro-
apoptotic signals
Once activated, these caspases cleave and
activate downstream effector caspases (including
caspase-3, -6, and -7), which in turn execute
apoptosis by cleaving cellular proteins

19. Recombinant LACTAPTIN
• To fully weaponize the prowess of lactaptin; RL 1 & 2 recombinant analogues of the
peptide were created using recombinant DNA technology.
• Analogs were produced in E.coli
• This may lead large scale industrial production of lactaptin without having to isolate
casein from animals
• RL2 has been used successfully, with a similar sequence to lactaptin, it is well embraced by the
human body. RL2 constitutively suppressed bcl-2 mRNA expression and down regulated Bcl-2
protein expression.
• RL2 contains the complete amino acid sequence of lactaptin (57–134 K-casein sequence) and
corresponding to 23–157 of human κ-casein
• The amino acids sequence of RL2 contains only one cysteine residue, which corresponds to Cys30 of
human κ-casein and responsible for the formation of disulphide bonds.
• RL2 is capable of binding α/β-tubulin and α-actinin-1 (Olga et al., 2014)
• RL2 penetrated the cells, interacted with cytoskeleton proteins and caused apoptosis via the
activation of caspases −3 and −7. RL2-dependent cell death was p53-independent and accompanied
by Bcl-2 depletion.
• RL1 was shown to induce only a small decrease in cell viability, whereas RL2 lowered the viability of
MCF-7 cells by 60% (Semenov et al., 2010).

20. CONCLUSION
Cancers remain a menace to humans despite tremendous efforts invested into
cancer therapy.
The molecular mechanisms of cancer development and the ways of their
regulation and control present a tedious task as far as the formulation of a
solution to cancer is concerned.
The studies of the mechanisms of apoptosis, whose disturbance is one of the
causes of malignant transformation of normal cells have established diverse
possibilities of inducing apoptosis of malignant cells.
Achieving apoptosis by tumor cell-specific protein factors may represent
modern anticancer drugs. Today, several proteins are used for human cancer
therapy
Studies have shown that Lactaptin is a very effective naturally-derived peptide
able to subject cancer cells to apoptosis and inhibit cancer’s survival
mechanisms like autophagy.
Lactaptin could therefore be a new molecule for the development of
anticancer drugs.

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