Pancreatic cancer is the eighth cancer leading cause of cancer-related death in the world and has a 5-year survival rate of 1–4% only. Gemcitabine is a first line agent for advanced pancreatic therapy; however, its efficacy is limited by its poor intracellular metabolism and chemoresistance. Studies have been conducted in an effort to improve gemcitabine treatment results by adding other chemotherapeutic agents, but none of them showed any significant advantage over gemcitabine monotherapy. We found that 85% of human pancreatic tumors analyzed by in situ hybridization analyses showed moderated to strong expression of the H19 gene. We designed a preclinical study combining gemcitabine treatment and a DNA-based therapy for pancreatic cancer using a non viral vector BC-819 (also known as DTA-H19), expressing the diphtheria toxin A chain under the control of the H19 gene regulatory sequences. The experiments conducted either in an orthotopic and heterotopic pancreatic carcinoma animal model showed better antitumor activity following the sequential administration of the vector BC-819 and gemcitabine as compared to the effect of each of them alone. The results presented in the current study indicate that treatment with BC-819 in combination with gemcitabine might be a viable new therapeutic option for patients with advanced pancreatic cancer.
In spite of the significant progress in treatment of different kinds of cancers, pancreatic cancer still has a very low rate of 5-year survival. The majority of patients with carcinoma of pancreas have already unresectable tumor and metastatic disease at presentation. A number of studies have been conducted in an effort to improve actual treatments by combining chemotherapeutic agents and radiotherapy, but none has yet shown more than a 10 month survival result [
Our group previously reported the use of a new DNA-based therapy for cancer treatment in which the plasmid BC-819 (also known as DTA-H19) drives the expression of diphtheria toxin A chain by the regulatory sequence of the H19 gene only into cancer cells [
This work was designed to demonstrate that a combination of effective local control of the tumor with systemic therapy can improve the results of the treatment.
The human ductal adenocarcinoma cell line CRL-1469 was obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA) and cultured in 90% DMEM-F12 medium and 10% fetal bovine serum (FBS). The hamster pancreatic ductal carcinoma cell line PC.1-0 was kindly provided by Dr. Buscail L. (Institut National de la Santé et de la Recherche Médicale U531, Institut Louis Bugnard, Institut Federatif de Recherche-31, Centre Hospitalier Universitaire Rangueil, Toulouse, France) and was cultured in 90% RPMI-1640 medium supplemented with 10% FBS. Antibiotic solution penicillin (180 units/mL), streptomycin (100
Gemcitabine (Eli Lilly) was diluted in saline.
The reporter plasmid Luc-H19 (which expresses the luciferase gene under the control of the H19 promoter) and the BC-819 construct were prepared as previously described [
The hamster model of pancreatic cancer was described in previous studies [
Animals were then sacrificed at day 18 at which time the tumors were measured and the abdominal cavity was searched for visible metastases. Tumors were excised, weighed, and their
The tumor volume was calculated as
The tumors were fixed in formalin, processed, and embedded in paraffin for pathology studies. All the tumors were histologically defined as pancreatic cancer.
The blood of 4 hamsters at each group was collected for the study of renal and liver function, blood counting (including CBC + differential, creatinine, calcium, phosphorus, urea, glucose, bilirubin, total protein, albumin, globulin, cholesterol, alkaline phosphatase, SGPT, SGOT, sodium, potassium, chloride).
Confluent CRL-1469 human pancreatic carcinoma cells were injected subcutaneously into the back of athymic nude mice (5-6-week-old and 20–30 grams) purchased from Harlan (Zeist, The Netherlands). Tumor-bearing mice were randomized when tumors reached approximately 6 mm diameter. After tumor development (30 days), three BC-819 administrations were performed, with a 2-day interval between each treatment, by direct injection into the tumor at days 0, 2, and 4. Treatment consisted of 25
To test survival, an additional xenograft model was used using pancreatic carcinoma cells from hamster [
All experiments were performed according to the rules of the Animal Ethics Committee.
As expected, gemcitabine treatment was effective in inhibiting the tumor growth as compared to the control group. However, the sequential use of BC-819 and gemcitabine demonstrated further advantages in reducing the tumor burden (Figures
Sequential administration of BC-819 and gemcitabine in a hamster orthotopic pancreatic carcinoma model. (a) Average of
Metastases occurrence was also analyzed: all animals in the control group showed multiple visible metastases at the end of the experiment, as compared to 63% of the animals treated with gemcitabine showing less than 4 metastases, and to 100% of the animals treated with BC-819 and gemcitabine showing no visible metastases (Table
Metastases observation developed in the hamster orthotopic pancreatic carcinoma model. Abdominal cavity was searched for metastases in the animals groups described in Figures
Treatment | No metastases | Few metastases (<4) | Numerous metastases (>4) |
---|---|---|---|
BC-819 + gemcitabine | 100% | ||
Gemcitabine | 37% | 63% | |
No treatment | 100% |
There was no evidence of toxicity in any treatment group based on animal appearance or weight loss. There were also no significant changes in hematology and chemistry in the BC-819 + gemcitabine treated group as compared to the untreated group.
In order to test the systemic toxicity of BC-819 given in combination with gemcitabine, several organ samples were collected from animals treated with BC-819 and gemcitabine at terminal histopathology examination. No gross or microscopic significant alterations were noted in the following organs: pancreas, liver, lung, bowel, spleen, heart, kidney, adrenal gland, lymph node, and gall bladder.
Histopathologic analysis showed that the sequential treatment with BC-819 and gemcitabine increased the necrotic area in the tumor in comparison with control groups (Figure
Histopathology analyses in orthotopically induced tumors in the pancreas of hamsters treated either with Luc-H19 + gemcitabine (a) or BC-819 + gemcitabine. (b) The arrows in pictures (a) and (b) mark the necrotic area (X10 original magnification for picture). An extensive area of necrotic tissue it is clearly shown in the BC-819-gemcitabine-treated tumor only. Slides were prepared from paraffin block sections and stained with hematoxylin eosin.
This study demonstrated that BC-819 used in sequential use with gemcitabine is more efficient than gemcitabine alone at controlling the tumor growth progression (reflected by lower tumors volume) and at preventing the occurrence of metastases.
Local human pancreatic tumor growth experiments were carried out in a subcutaneous xenograft model. This animal model has proven useful in the measurement of tumor progression and has been reliable and reproducible.
Sequential use of BC-819 and gemcitabine resulted in a significant delay in tumor growth progression as compared to the other groups (Figure
Sequential administration of BC-819 and gemcitabine in a subcutaneous human pancreatic tumor in nude mouse model. and Confluent CRL-1469 human pancreatic carcinoma cells were injected subcutaneously into the back of athymic nude mice. After tumor development (30 days), three BC-819 administrations were performed, with a 2-day interval between each treatment, by direct injection into the tumor at days 0, 2, and 4. Mice were randomly divided into one of the following groups: control (glucose 5%), BC-819 alone, gemcitabine alone and BC-819 + gemcitabine. Average tumor growth progression of control (
A significant difference in tumor growth progression was found between the group of BC-819 + gemcitabine versus gemcitabine alone (
Both groups treated with gemcitabine alone or sequentially with BC-819 showed similar weight loss (up to 20%) as compared to the control group or the group treated with BC-819 alone (data not shown).
Local tumor growth and survival experiments were carried out in a subcutaneous xenograft model.
Sequential use of BC-819 and gemcitabine resulted in a significant difference in tumor growth progression as compared to the other treatment groups (Figure
The effect of sequential administration of BC-819 and gemcitabine on tumor progression and on the survival of a nude mice heterotopic pancreatic carcinoma model. Pancreatic carcinoma cells from hamster (PC1-0 cells) were injected subcutaneously into the back of athymic nude mice. After tumor development, 3 treatments of plasmid vectors were given, with a 2-day interval between each treatment, by direct injection into the tumor. Mice were randomly divided into 4 groups of 4 animals each and received the following treatments: Luc-H19 (control vector), BC-819, Luc-H19 + gemcitabine, BC-819 + gemcitabine. Mice were sacrificed when tumor reached a diameter larger than 13 mm. (a) Average tumor growth progression comparing the tumor volume measured after the last treatment to the volume measured before the first treatment. (b) Percentage of mice with a tumor diameter <13 mm as a function of time after the start of the treatment. The days or treatment are marked by arrows.
21 days after the first treatment the survival rate in the group of mice treated with both BC-819 and gemcitabine was 100% while in the group treated with BC-819 alone was 50% and 0% in the groups treated with either the combination of gemcitabine and Luc-H19 or Luc-H19 alone (Figure
A significant difference in tumor growth progression was found between the group of BC-819 + gemcitabine versus Luc-H19 + gemcitabine (
The results show a significant increase in survival of the group treated with BC-819 + gemcitabine versus Luc-H19 + gemcitabine or BC-819 alone (Figure
Along with moderate differences in the tumor progression between the mice treated with BC-819 alone and those treated with the combination of BC-819 and gemcitabine, survival in the last group was also significantly longer.
The present study proposes a successful approach for the treatment of pancreatic cancer combining conventional chemotherapy used nowadays and a DNA-based therapy expressing a toxin under the control of regulatory sequences of a differentially expressed gene, the H19 gene. The human H19 gene is an imprinted gene, expressed from the maternal allele in several tissues during embryo development and repressed right after birth [
However, this treatment delaying tumor growth may not efficiently prevent metastatic spread. The combination of local therapy with systemic chemotherapeutic agent is very likely to be more effective in the control of the disease [
To date, some gemcitabine-containing combinations have demonstrated modest improvement when compared to gemcitabine alone, particularly in patients with good performance status [
This work was supported by BioCancell Therapeutics Israel Ltd.