Salvestrols

and CYP1B1 A natural team fighting cancer naturally

Professor Dan Burke

Salvestrol Natural Products, Leicester UK

1995
UNIVERSITY OF ABERDEEN

CYP1B1
Enzyme protein very highly over-expressed in cancer cells (human biopsies /

immunohistochemistry)
Undetectable in most normal cells ... trace levels in some

normal cells stained purple O CYP1B1 cancer cells stained black P CYP1B1

oesophageal cancer
Murray, Melvin, Greenlee & Burke; Annu. Rev. Pharmacol. Toxicol. 41: 297-316 (2001)

Expression of CYPIBI protein in 14 different types of cancer and normal tissue

Tissue Bladder Brain Breast Colon Connective tissue Esophagus Kidney Liver Lung Lymph node Ovary Skin Small intestine Stomach Testis Uterus Normal (# positive / # tested) 0/8 0/12 0/10 0/10 0/9 0/9 0/11 0/8 0/8 0/5 0/5 0/6 0/5 0/10 0/8 0/7 Cancer (# positive / # tested) 8/8 11/l2 12/12 11/12 8/9 8/8 11/11 Not tested 7/8 9/9 7/7 6/6 Not tested 9/10 8/8 7/7 (transitional cell carcinoma) (astrocytoma) (invasive ductal carcinoma) (adenocarcinoma) (sarcoma) (squamous carcinoma) (carcinoma) (squamous carcinoma) (non-Hodgkin's lymphoma) (adenocarcinoma) (squamous carcinoma) (adenocarcinoma) (malignant germ cell tumor) (adenocarcinoma)

Murray, Taylor, McFadyen, McKay, Greenlee, Burke & Melvin; Cancer Res. 57: 3026-31031 (1997)

Expression of CYPIBI protein in 14 different types of cancer and normal tissue

Tissue Bladder Brain Breast Colon Connective tissue Esophagus Kidney Liver Lung Lymph node Ovary Skin Small intestine Stomach Testis Uterus Normal (# positive / # tested) 0/8 0/12 0/10 0/10 0/9 0/9 0/11 0/8 0/8 0/5 0/5 0/6 0/5 0/10 0/8 0/7 Cancer (# positive / # tested) 8/8 11/l2 12/12 11/12 8/9 8/8 11/11 Not tested 7/8 9/9 7/7 6/6 Not tested 9/10 8/8 7/7 (transitional cell carcinoma) (astrocytoma) (invasive ductal carcinoma) (adenocarcinoma) (sarcoma) (squamous carcinoma) (carcinoma) (squamous carcinoma) (non-Hodgkin's lymphoma) (adenocarcinoma) (squamous carcinoma) (adenocarcinoma) (malignant germ cell tumor) (adenocarcinoma)

Murray, Taylor, McFadyen, McKay, Greenlee, Burke & Melvin; Cancer Res. 57: 3026-31031 (1997)

Expression of CYPIBI protein in 14 different types of cancer and normal tissue

Tissue Bladder Brain Breast Colon Connective tissue Esophagus Kidney Liver Lung Lymph node Ovary Skin Small intestine Stomach Testis Uterus Normal (# positive / # tested) 0/8 0/12 0/10 0/10 0/9 0/9 0/11 0/8 0/8 0/5 0/5 0/6 0/5 0/10 0/8 0/7 Cancer (# positive / # tested) 8/8 11/l2 12/12 11/12 8/9 8/8 11/11 Not tested 7/8 9/9 7/7 6/6 Not tested 9/10 8/8 7/7 (transitional cell carcinoma) (astrocytoma) (invasive ductal carcinoma) (adenocarcinoma) (sarcoma) (squamous carcinoma) (carcinoma) (squamous carcinoma) (non-Hodgkin's lymphoma) (adenocarcinoma) (squamous carcinoma) (adenocarcinoma) (malignant germ cell tumor) (adenocarcinoma)

Total

0 / 130 (0%)

122 / 127 (96%)

Murray, Taylor, McFadyen, McKay, Greenlee, Burke & Melvin; Cancer Res. 57: 3026-31031 (1997)

cancer cells stained brown

PCYP1B1
normal cells stained blue OCYP1B1
:

all cells blue:

O CYP1B1

prostate carcinoma

normal prostate

Carnell, Smith, Daley et al.; Int. J. Radiation Oncology Biol. Phys. 58: 500-509 (2004)

CYP1B1 protein is highly overexpressed in all types of cancer* and in metastases and precancer cells

Bladder Brain Breast Cervix Colon Connective tissue

Head & Neck Kidney Leukaemia Lung Lymphoma Melanoma Mesothelioma

Myeloma Ovary Oesophagus

Prostate
Stomach Testis Uterus

* confirmed by Dana-Farber Cancer Institute, Boston, USA

CYP1B1 protein expression controlled by microRNA 1 and untranslated region DNA interactions 2
CYP1B1 gene ribosome
1. Tsuchiya, Y. et al., Cancer Res. 66: 9090-98 (2006) 2. Devlin, A. et al., Mol. Carcin. 49: 190-99 (2010)

normal cell
CYP1B1 mRNA but no CYP1B1 protein

nucleus
mRNA

CYP1B1 protein

DNA CYP1B1 gene nucleus

mRNA

interactions RNA
ribosome

cancer cell cancer cell

CYP1B1 mRNA and CYP1B1 protein

normal cell

Lung cancer patient CYP1B1 peptide level CYP1B1 as imaging tool for cancer in surgery CYP1B1 in plasma as cancer biomarker plasma sample no.
0016005001 0016003001 0016005002 0016001001 0016002002 0016003002 0016004001 0016002001 0016004002 0016006002 0016009001 0016006001 0016009002 0016001002 0016008002 0016007001 0016007002 0016008001

x higher than pooled normal plasma

6291 1234 1207 799 279 276 222 214 162 148 135 134 124 121 119 118 97 93

CYP1B1 peptide measured in plasma using SISCAPA absolute quantitation mass spectrometry CYP1B1 in pooled normal plasma: < 10 amol/l inactive

 The majority of anticancer drugs are not truly selective for cancer cells.

Their efficacy is limited by the damage they also

cause to normal cells.
Prof. Bill Denny, University of Auckland, NZ Cancer Investigation 22: 604-619 (2004)
---------------------------------------------------------------------------------------------

The anticancer drug targets not just in cancer cells but also in many critically important normal cells

1995
UNIVERSITY OF ABERDEEN

CYP1B1
Protein present virtually only in cancer cells Cytochrome P450 enzyme that can alter chemical structure of drugs

Change drug's structure ... change its effects

Activate prodrug new

type of anticancer drug

Gerry Potter
Professor of Medicinal Chemistry

To design anticancer prodrug Designed anticancer prodrug

activated by CYP1B1 to be activated by CYP1B1 Pharmacophore Theorized also in natural phytochemical compounds e.g. in oranges 2002 -

had invented Abiraterone

SALVESTROLS

Salvestrols
natural phytochemical anticancer prodrugs 2002 15+ years university research
Unique mechanism of action Activated by CYP1B1 in cancer cells Stimulate apoptosis Kill cancer cells without harming normal cells In fruits, vegetables and herbs in diet Historically Salvestrols probably helped protect against cancer Now grossly deficient in diet

Salvestrols
natural anticancer prodrugs
no CYP1B1
drug target

initially

inactive
and innocuous

CYP1B1

enzyme only in cancer cells

active
Salvestrol metabolite
drug target

not activated

unharmed
normal cell

cancer cell

Sources of Salvestrols Natural Sources of Salvestrols

FRUIT Apples Blackcurrants Cranberries VEGETABLES Avocados HERBS Artichoke

Brussels Sprouts
Capsicums Cabbage

Basil
Dandelion Milk Thistle

Grapes
Oranges Strawberries

Cauliflower
Olives

Parsley
Thyme

Dose-response curve for a Salvestrol with human cells in culture

Salvestrols kill % cells killed at each dose cancer cells while 100 sparing normal cells
75 50 25 0 0.1 1 10 Salvestrol dose ( M ) Log dose scale
mean sem

cancer cells have CYP1B1 killed : higher dose more killed normal cells lack CYP1B1 unharmed : at any dose

Salvestrol dose-response curve with specific chemical inhibitor of CYP1B1

Tan H. et al. J. Pharm. Pharmacol. 59: S158 (2007)

ability of % cells killed at each dose Salvestrols to kill 100 cancer cells while sparing normal 75 cells is due to CYP1B1 50 in cancer cells
25 0

plus CYP1B1 inhibitor cancer cells without inhibitor cancer cells with inhibitor normal cells not killed when CYP1B1 is blocked

0.1 1 10 Salvestrol dose ( M )

mean sem

Dose-response curve for a Salvestrol with human cells in culture

Salvestrol with % cells killed at each dose high potency and 100 very high selectivity
75 50 25 0

breast cancer cancer cells have CYP1B1 breast noncancer cells lack CYP1B1
28.000 M
0.1 1.0 10 100

therapeutic index
= 25,000

0.0011 M
0.0001 0.001 0.01

mean sem

Salvestrol dose ( M )

Cancer cells treated with Salvestrol

cell succumbing to Salvestrol
nucleus

cytoplasm

cancer cells not yet affected

Cancer cells treated with Salvestrol

condensed DNA ... cell death by apoptosis

DNA (blue)

CYP enzyme (red)

tyrosine kinase inhibition apoptosis stimulation not antioxidant

Salvestrols should contribute to

protection
by fruit & vegetables

against cancer

Salvestrols enough bioavailability for protection

20 x Palaeolithic daily diet level as 2x daily supplement : used by many cancer patients
50% 3.5 x Salvestrol blood level Palaeolithic

10% cancer cell kill

daily diet level as 1x daily supplement

12

8 am

time of day (h)

24

36

8 am next day

Salvestrols should contribute to

protection
by fruit & vegetables against cancer but

Modern diet very deficient in Salvestrols

Why?

Main reason for Salvestrol deficiency Agricultural spraying with synthetic fungicides Salvestrols ... phytoalexins ... ... produced in response to fungal attack Spraying eliminates all fungi ... ... removes stimulus for Salvestrol synthesis Organic has 30x more Salvestrols

Other reasons for Salvestrol deficiency Salvestrols taste bitter

Debittering of manufactured fruit juices

Modern sweeter varieties of fruit

Salvestrols readily dissolve in boiling water

Boiling vegetables extensively in lots of water

Solution to problem - Salvestrol supplement

Salvestrols and dogs

CYP1B1 in dogs

Amino acid sequence 84% similar to human

Over-expressed in dog cancer ? ... don't know yet Dog drug metabolism pathways similar to human Expect Salvestrols to be bioavailable & metabolized in dogs similar to in humans Expect Salvestrols to be anticancer prodrugs in dogs as in humans

 Mrs K : standard poodle - lower leg tumour predicted 4 weeks to live given Salvestrol supplement for 7 months - although leg eventually amputated, cancer did not spread and dog remained bright and active vet said to continue with Salvestrols because they prevented a normally very aggressive type of cancer from spreading beyond the leg. YR : dog had bladder cancer given Salvestrol supplement for some months - cancer no longer detectable. DB : dog has liver cancer being given Salvestrol supplement daily

" doing well " at this point.

MM : dog has sarcoma predicted 6-12 months to live being given Salvestrol supplement daily initially kept having to have surgery but after Salvestrols tumours reduced from fist size to size of Canadian dollar coin (2.6 cm dia.) - so at six month check up no surgery required.

Thank You
for listening