. This study suggests that simultaneous treatment of brucine and gemcitabine at the same time exhibits best synergistic/additive effect in human breast cancer cells. .Targeted drug delivery systems are needed to take advantage of drugs like brucine, in view of its cytotoxic nature.

1. Dr. Mamatha Serasanambati, Ph.D
Department of Biochemistry
Sri Venkateswara University
Tirupati
Anticancer Efficacy of Brucine and
Gemcitabine Combination in Human
Breast Cancer Cell Lines

2.  Introduction
 Objectives
 Materials and Methods
 Results
 Conclusions
Contents

3. Cancer is the price the higher organisms pay
for having the facility of ‘differentiation /
organ formation’
Ability of these cells to divide, invade and
multiply anywhere in the body forming tumors
Benign tumors - non-cancerous
Malignant tumors - cancerous tumors, these
tumors are usually rapidly growing increasing
size of tumours
Metastasis - Cells detach from the primary
tumor and move to other parts of the body
where they form new malignant tumors
Cancer
ZYGOTE
D I F F E R E N T I A TI O N
ORGAN FORMATION
De-Differentiation
Or Cancer

4. Breast cancer
Breast cancer is the most common type of cancer
among women
Breast cancer starts when certain cells of the breast
grow out of control
As the cells grow, they may form lumps or tumors
Lumps can either be benign or malignant
Malignant growths are called breast cancer

5. Worldwide Cancer Incidence &
Breast Cancer Position
 Of all the estimated 141 million adults
world wide diagnosed with cancer,
1.67 millions were identified to be
with breast cancer, the 2nd largest behind
lung cancer (WHO, 2012)
 Breast cancer is the most common
cancer and accounts for 25 - 31% of all
cancers in women in India
(Source: Population Based Cancer Registries
2009 -2011. ICMR)

6. Risk factors
Factors that cannot be prevented
Gender, Aging, Menstrual cycle, Family History
Genetic Risk Factors
BRCA1 50% – 85%
BRCA2 40% – 80%
TP53 < 1%
Factors that can be prevented
Oral contraceptive use, Birth control, Breast feeding, Alcohol , Obesity
Physical inactivity, Smoking, Environmental Factors
Stages
Staging is described by the TNM system
Stage 0 , Stage I
Stage II , Stage III
Stage IV
Diagnosis / Screening
Breast self-exam
Clinical breast exam
Annual mammogram

7. Treatment
Treatment depends on stage of the tumor
More than one treatment may be necessary
Surgery
Radiation Therapy
Chemo Theraphy
Hormone Theraphy: It showed initial promise,
but later looks like leading to ovarian cancer.

8.  Natural plant alkaloid
 Strychnos nux-vomica seeds
 Brucine is cytotoxic though it was used in liver cancer in
Chinese medicine (Bensky et al, 1986)
 Brucine showed anti-proliferative effects in different cancer cell lines such as
HeLa , K562 (Cai et al, 1998), HepG2 (Deng et al, 2006), SMMC-7221
(Yin et al, 2007) and multiple myeloma cell lines (Rao et al, 2009)
 It arrests the cell cycle at G1 phase in colon cancer cells (Zheng et al, 2013)
 In non-small cell lung cancer, it inhibits the NF-kB activity (Zhu et al,
2012)
 Other Actions
• In addition, it is an antioxidant, anti-inflammatory and anti-diabetic
Brucine

9. • Difluorodeoxycytidine (dFdCyd)
• First approved by FDA in 1996 for the treatment of breast cancer
• In 2006, it was further approved for ovarian cancer
• Recently approved for treatment of pancreatic cancer
• Chemotherapeutic agent
 Treat various types of cancer
• Non small cell lung cancer, Pancreatic cancer,
Ovarian cancer and Breast cancer
 Combination Therapy with
• Cisplatin for non small cell lung cancer (Sandler et al, 2000)
• Curcumin for Pancreatic cancer (Kunnumakkara et al, 2007) and Bladder cancer
(Tharakan et al, 2010)
 Major drawback of gemcitabine in monotherapy is Chemoresistance, as it activates
NF-kB
Gemcitabine

10. Mechanism of action
Fluoro-Cytidine
(derivative of Gemcitabine)
Blocks DNA synthesis
 During semi-conservative DNA replication, normally, A pairs with T and G with C
 But, derivative of gemcitabine pairs with guanine as a fraudulent base, instead of
regular cytosine and results in termination of DNA chain progression

11.  In combination, the drugs may increase either the efficacy of treatment
and/or the chance of lowering adverse effects due to the use of individual
ones at lower concentrations when combined together
 To identify new agents
 Natural products like brucine in combination with anticancer agents like
gemcitabine
 Combination efficacy of brucine and gemcitabine was NOT tested before
Advantages of Combination Chemotherapy

12.  Three of human breast cancer cell lines: MCF-7, MDA MB - 231 and - 436
 All the three cell lines are negative for human epidermal growth factor receptor
(HER2-)
 MCF-7 cell line is invasive ductal carcinoma (IDC) type - ER+ , PR+ and to
express wild type tumor repressor protein p53 (TP53+), originally isolated in and
named after Michigan Cancer Foundation (MCF), USA
 MDA MB-231 cell line is adenocarcinoma type - ER- , PR- and mutated TP53
 MDA MB-436 cell line is IDC type - ER- , PR- and no expression of TP53
which were originally established in MD Anderson Cancer Research Center as
Metastatic Breast cancer cell lines, hence, designated as MDA MB series
Rationale behind the choice of Breast cancer cell lines

13. Objectives
 Testing anticancer activity of brucine and gemcitabine
combination in MCF-7 (IDC) cell line
 Combination effect of brucine and gemcitabine in two basal B
type of human breast cancer cell lines namely,
 MDA MB-231 (adenocarcinoma)
 MDA MB-436 (IDC)

14.  Materials
Cell lines
Human breast cancer cell lines: MCF-7, MDA MB-231 and -436 were
purchased from ATCC, Manassas, VA , USA
 Assays/Analyses to be performed
 MTT Assay (Cell viability/Cytotoxicity/Anti-Proliferation)
 Flow Cytometry (Cell cycle distribution analysis)
 Western blot analysis (NF-kB expression analysis)
 Soft agar colony formation assay (anchorage independent growth)
 In-vitro scratch assay (cell migration/metastasis)
Materials and Methods

15. Dose response and time course effect of brucine and gemcitabine
 Brucine (µM) : 0, 1, 10, 50, 100, 150, 200, 250 & 300
 Gemcitabine (µM) : 0, 0.01, 0.05, 0.1, 1, 10, 25, 50 & 100
 Time course (h) : 24 & 48
 Breast Cancer Cell Lines : MCF-7, MDA MB-231 & MDA MB-436
Cell Viability/
Cytotoxicity /
Anti-Proliferation Test : MTT Assay
Results

16. Principle of the assay:
 It is a colorimetric assay, in which MTT dye is
taken up by the living cells and mitochondrial
succinate dehydrogenase present in the cell reduces
the yellow colored MTT dye to an insoluble, dark
purple coloured formazan product (570 nm)
 Since reduction of MTT can only occur in
metabolically active cells the level of activity
is a measure of viability of the cells
 Hence, the difference in the viabilities between
untreated and drug treated cells was calculated
as anti-proliferative effect
MTT Assay

17. Figure 1: The maximum inhibitions noticed at the concentration of
A: Brucine (300 µM) -76%
B: Gemcitabine (100 µM) -57% . *p ≤ 0.001 (n=3)
Anti-proliferative Effect: MCF-7

18. Figure 2: The maximum inhibitions noticed at the concentration of
A: Brucine (300 µM) - 71%
B: Gemcitabine (100 µM) - 53% . *p ≤ 0.001 (n=3)
Anti-proliferative Effect: MDA MB-231

19. Figure 3: The maximum inhibitions noticed at the concentration of
A: Brucine (300 µM) - 73%
B: Gemcitabine (150 µM) - 54%. *p ≤ 0.001 (n=3)
Anti-proliferative Effect: MDA MB-436

20. Drugs MCF - 7 MDA MB - 231 MDA MB - 436
Brucine 100 µM 116.3 µM 123.8 µM
Gemcitabine 68.4 µM 70 µM 79.8 µM
IC50 Values
MCF-7 < MDA MB - 231 < MDA MB - 436

21.  Brucine (μM) : 50 100 300
 Gemcitabine (μM) : 25 50 100 25 50 100 25 50 100
were performed in three formats
 First : Cells were treated with brucine and gemcitabine at the same time
 Second: Cells were pre-treated with gemcitabine for 12 h and then exposed to
brucine
 Third: Cells were pre-treated with brucine for 12 h and then exposed to
gemcitabine
Cell viability/Cytotoxicity/Anti-Proliferation Test : MTT at 570 nm
Format of combination studies

22. Combination Effect on Anti-proliferation in MCF-7
Figure 4: Combination treatment
with brucine (300 µM) and
gemcitabine (100 µM) produced
maximum inhibition of cell
proliferation (at 48 h)
A: 81%; B: 79% & C: 69%
*p ≤ 0.001 (n=3)

23. Figure 5: Combination treatment
with brucine (300 µM) and
gemcitabine (100 µM) produced
maximum inhibition of cell
proliferation (at 48 h) by A: 80%;
B: 77% & C:63%
*p ≤ 0.001 (n=3)
Combination Effect on Anti-proliferation in MDA MB-231

24. Combination Effect on Anti-proliferation in MDA MB-436
Figure 6: Combination treatment with
brucine (300 µM) and gemcitabine
(100 µM) produced maximum
inhibition of cell proliferation (at 48 h)
A: 68%; B: 66% & C: 59%
*p ≤ 0.001 (n=3)

25. Conceptually, Synergism is more than additive effect; Antagonism is less than additive effect
Determining Synergism using CompuSyn Software
<1 Synergistic +++++
= 1 Additive +++
˃1 Antagonist - - -

26. Combination effect on Anti-Proliferative Activity
B +G (µM) CI Effect
300+100 0.12 Strongly Synergistic
300+50 0.28 Strongly Synergistic
300+25 0.38 synergistic
B +G (µM) CI Effect
300+100 0.16 Strongly Synergistic
300+50 0.40 Synergistic
300+25 0.60 synergistic
MCF-7 MDA MB-231

27. B +G (µM) CI Effect
300+100 1.06 Nearly Additive
Combination effect on Inhibition of Proliferation
Source: CompuSyn - Software
(Drug Combinations and
for General Dose-Effect Analysis)
http://www.combosyn.com/feature.html
MDA MB-436

28. Cell line
% of inhibition of cell proliferation
at the combination of brucine 300 µM + gemcitabine 100 µM at 48 h
Treat at same time
Pre-treat with gemcitabine
followed by brucine
Pre-treat with brucine
followed by gemcitabine
MCF-7 81 79 69
MDA MB-231 80 77 63
MDA MB-436 68 66 59
Summary of combination studies
Summary of the combination studies in all three cell lines and in three formats
 In the format of pre-treatment with brucine, followed by gemcitabine produced the least
% inhibition in all the cell lines,
 Of all the 3 cell lines, MDA MB-436 produced the least % inhibition by drug
combination
 Hence, further studies were carried out with MCF-7 & MDA MB-231 cells treated with
both drugs at the same time

29. Human Breast Cancer Cells: BRCA 1/2
 Mutated BRCA1: MDA-MB-436
and no expression of TP53
 Non-mutated BRCA1/2 :
• MCF-7
• MDA MB – 231
 Mutations in BRCA1 and BRCA2 genes
are associated with a high lifetime risk of
breast and ovarian cancers.
 BRCA1 and BRCA2 proteins play a major
role repair of DNA following double-strand
breaks

30. Cell Cycle
 Distribution of Cell Cycle analysis was carried out for MCF-7 & MDA
MB-231 cells in the format of treating with both drugs at the same time
with B+G =300+100 µM

31.  One of the applications of Flow Cytometry
• DNA & Cell cycle distribution
• 2n amount DNA (diploid) - G1 phase
• 4n amount DNA (tetraploid) - G2 phase
• In between 2n and 4n amount - S phase
 Principle
• Flow cytometry involves the analysis of the fluorescence and light
scatter properties of single particles (e.g. cells, nuclei, chromosomes)
during their passage with in a narrow, precisely defined liquid stream
Flow cytometry
Analyzing Cell Cycle Distribution
Excitation maximum 493nm; Emission maximum 636nm
G1
S
G2/M

32. Cell cycle analysis in MCF-7 cells
1.6x; 1.7x and 2.1x fold increase in G1 phase ; 2.3x; 2x and 1.5x fold increase in S phase
3.1x; 3.4x and 17.2x fold decrease in G2 phase. *p ≤ 0.001 (n=3)
Group %G1 %S %G2
Untreated Control 57.0±0.7 19.3±0.7 23.7±1.2
B 300 µM 60.1±1.8* 23.5±4.1* 16.4±3.2*
G 100 µM 64.0±1.5* 20.9±1.1 15.1±0.7*
B + G 300+100 µM 81.3±0.8* 15.8±1.4* 3.0±1.4*

33. Cell cycle analysis in MDA MB-231 cells
Group %G1 %S %G2
Untreated Control 41.2±0.9 46.8±1 12.0±0.9
B 300 µM 52.1±0.9* 18.1±3.2* 29.9±5.6*
G 100 µM 57.3±1.2* 14.4±1.3* 28.2±2.8*
B + G 300+100 µM 70.1±0.5* 10.1±0.5* 19.9±1.6*
1.3x; 1.4x and 1.7x fold increase in G1phase ; 2.6x; 3.2x and 4.7x fold decrease in S phase
2.5x; 2.4x and 1.7x fold increase in G2 phase. *p ≤ 0.001 (n=3)

34. Mechanism of TP53 Translocation between Nucleus and
Cytoplasm
 In normal cells, p53 is transported from nucleus to cytoplasm and performs tumor
suppressor function
 However, when cells are arrested at G1/S phase, p53 is relocated to nucleus from
cytoplasm and results in cell death (apoptosis)
Source: Liang and Clarke 2001. Eur J Biochem 268: 2779-83

35. Figure 9:
• Brucine (300 µM) inhibited the expression of NF-kB subunit (p65) significantly up to 83%
• 100 µM gemcitabine was least effective 16%
• In combination treatment NF-kB expression (p65) was significantly inhibited up to 73%
• *p≤ 0.001(n=3)
NF-kB expression in MCF-7
A

36. Figure 10:
• Brucine inhibited the expression of NF-kB subunit (p65) significantly at 300 µM up to 89%
• 100 µM gemcitabine was least effective - 9%
• In combination treatment NF-kB expression (p65) was significantly inhibited up to 69%
• *p≤ 0.001 (n=3)
NF-kB expression in MDA MB-231
NF kB (p65)
A

37. Combination effect of brucine and gemcitabine in breast cancer-
Proposed Mechanism of action regarding NF-kB
In combination therapy, brucine compensates for the drug resistance of gemcitabine
Brucine
NF - kB
Gemcitabine
Activation
Inhibition

38.  Hence, soft agar colony formation
is an anchorage independent
growth assay in soft agar, which
is considered the most stringent
assay for determining malignant
status of cells
Soft agar colony formation assay
Normal cells Tumor cells
 Require anchorage  Not require
 Exhibit contact inhibition  Loss of contact inhibition
 Cells don’t grow over one another  Cells can grow over one another
Normal cells
Colonies

39. Figure 11:
• Brucine (100 µM) caused inhibition of colony formation - 42%.
• Gemcitabine (50 µM) also resulted in inhibition of colony formation - 41%
• Combination treatment inhibits - at 50+50 µM: 64% ; 100+25 µM: 78% and 100+50 µM: 80 %
• *p≤ 0.005 (n=3)
Inhibition of anchorage independent growth in MCF-7

40. Figure 12:
• Brucine (100 µM) caused slightly higher inhibition of colony formation - 30%
• Gemcitabine (50 µM) also resulted in inhibition of colony formation - 26%
• In combination treatment reduce at 50+50 µM: 64% ; 100+25 µM :78% and 100+50 µM: 79 %
• *p ≤ 0.001 (n=3)
Inhibition of anchorage independent growth in MDA MB-231

41. Combination effect of colony formation
B +G (µM) CI Effect
100+25 0.45 Synergistic
100+50 0.53 Synergistic
50+25 1.1 Antagonistic
50+50 1.3 Antagonistic
B +G (µM) CI Effect
100 + 25 0.44 Synergistic
100+ 50 0.62 Synergistic
50+25 0.98 Near Additive
50+50 0.99 Additive
MCF-7 MDA MB-231

42.  The in vitro scratch assay is an easy, low-cost and well- developed
method to measure cell migration in vitro
 The basic steps involved creating a "scratch" in a cell monolayer,
capturing the images a the beginning and at regular intervals during cell
migration to close the scratch, and comparing the images to quantify the
migration rate of the cells
 Compared to other methods, the in vitro scratch assay is particularly
suitable for studies on the effects of cell-matrix and cell-cell interactions
on cell migration, mimic cell migration in vivo, which in general occurs
during cancer metastasis
 In the current context, the cell migration assay indicates the metastatic
nature of the cancer cells
In vitro scratch assay
Zero h - Scratch
48 h –Ctrl
48 h – Drug treated
Wimasis image analysis, Source: www.wimasis.com

43. Figure 13:
• Brucine at 100 µM reduced a maximum of cell migration by 13%
• Gemcitabine at 50 µM reduced a maximum of cell migration by 23%
• In combination treatment of both brucine (100 µM) and gemcitabine (50 µM) - up to 70 %
• *p≤ 0.001 (n=3)
Inhibits cell migration in MCF-7
b

44. Figure 14:
• Brucine (100 µM) reduced a maximum of cell migration by 16 %
• Gemcitabine (50 µM) - 20%
• In combination treatment of both brucine (100 µM) and gemcitabine (50 µM) - up to 62%
• *p ≤ 0.001 (n=3)
Inhibits cell migration in MDA MB-231
b

45. Combination effects of Brucine and Gemcitabine
on Cell Migration
B+G (µM) CI Effect
50+25 0.81 Synergistic
100+25 0.99 Nearly Additive
100+50 1.5 Antagonistic
50+50 1.3 Antagonistic
B+G (µM) CI Effect
50+25 0.91 Slightly Synergistic
50+50 1.4 Antagonistic
100+25 1.2 Antagonistic
100+50 1.8 Antagonistic
MCF-7 MDA MB-231

46.  The results in the present study indicated that brucine in combination with gemcitabine
significantly decreased cell proliferation both in dose and time dependent manner in
human breast cancer cells, in the order of MCF-7 > MDA MB-231 > MDA MB-436
 The experimental evidence from this study suggests that simultaneous treatment of
brucine and gemcitabine at the same time exhibits best synergistic/additive effect in
human breast cancer cells
 Though quantitatively similar inhibitions were observed in MCF-7 & MDA MB-231
cells, there was a qualitative difference with regards to cell cycle analysis
 Brucine and gemcitabine combination resulted in increased accumulation of cells in G1
phase for both the cell lines studied (MCF-7 and MDA MB-231). However, while an
increase in S and decrease in G2 phase cells was observed in MCF-7 cells, it was the
other way in MDA MB-231 cells
Conclusions

47.  Brucine alone or in combination only suppressed NF-kB expression in both MCF-7 &
MDA MB-231 cells, compensating for the lack of such ability by gemcitabine
 The combination of brucine and gemcitabine resulted in synergistic and additive
inhibitory effect on colony formation (a property of malignancy) and cell migration
(a property of metastasis) in both MCF-7 and MDA MB-231 cells
 Hence, we consider combined treatment of brucine and gemcitabine could be a
potential anti-cancer agent in breast cancer cells
 However, in-depth studies need to be conducted to confirm its efficacy in animal
models & clinical trials
 Also, targeted drug delivery systems are needed to take advantage of drugs like
brucine, in view of its cytotoxic nature
Conclusions ….contd

48. PUBLICATIONS
 Mamatha. Serasanambati, . . . ., Damodar Reddy. Chilakapati. Gemcitabine
and brucine inhibit MDA MB-436 human breast cancer cells, Sch. Acad. J.
Biosci., 2014;2(2): 71-75
 Mamatha. Serasanambati, . . . . , Damodar Reddy. Chilakapati. Anti-cancer
effects of brucine and gemcitabine combination in MCF-7 human breast
cancer cells. Natural Product Research, 2014; DOI: 10.1080/14786419.
2014.951932 (IF-1.2)
 Mamatha. Serasanambati, . . . ., Damodar Reddy. Chilakapati. Inhibitory
effect of gemcitabine and brucine on MDA MB-231human breast cancer cells.
International Journal of Drug Delivery, 2014; 6(2) (IF-1.3)