Israeli researchers founds a small molecule known as PJ34 that induce the self-destruction of pancreatic cancer as much as 90 percent a month

A new treatment developed by Tel Aviv University could induce the destruction of pancreatic cancer cells, eradicating the number of cancerous cells by up to 90% after two weeks of daily injections of a small molecule known as PJ34.

Pancreatic cancer is one of the hardest cancers to treat. Most people who are diagnosed with the disease do not even live five years after being diagnosed.

The paper was published in the October issue of the journal Oncotarget, a peer-reviewed biomedical journal that covers oncology research.

The journal is a lesser-known publication within the academic world, and the studies it publishes generally have a lower impact factor than those published in leading medical journals like The New England Journal of Medicine, Nature or The Lancet.

(Impact factor is the number of times a published article has been cited in a year, divided by the total number of articles published in the previous two years.)

The study, led by Prof. Malka Cohen-Armon and her team at TAU’s Sackler Faculty of Medicine, in collaboration with Dr. Talia Golan’s team at the Cancer Research Center at Sheba Medical Center, was recently published in the journal Oncotarget.Specifically, the study found that PJ34, when injected intravenously, causes the self-destruction of human cancer cells during mitosis, the scientific term for cell division.

The research was conducted with xenografts, transplantation of human pancreatic cancer into immunocompromised mice.

A month after being injected with the molecule daily for 14 days, “there was a reduction of 90% of pancreatic cells in the tumor,” Cohen-Armon told The Jerusalem Post. “In one mouse, the tumor completely disappeared.”

“This molecule causes an anomaly during mitosis of human cancer cells, provoking rapid cell death,” she said.

“Thus, cell multiplication itself resulted in cell death in the treated cancer cells.”Moreover, she said, PJ34 appears to have no impact on healthy cells, thus “no adverse effects were observed.” The mice, she said, continued to grow and gain weight as usual.

Pancreatic cancer is currently resistant to all treatments, and patients have poor chances of surviving for five years after being diagnosed.

Prof. Malka Cohen-Armon, left, of Tel Aviv University and Dr. Talia Golan of Sheba Medical Center (Tel- Aviv University).

In parallel studies, the research team found that the molecule “acts efficiently” when they tested it on other types of deadly cancer cell cultures in the lab, including aggressive forms of breast, lung, brain and ovarian cancer, all of which are resistant to current therapies, she said.

Cohen-Armon added that the team hopes to start testing the effect of the molecule on larger animals, and eventually on humans, which could take some two years, depending on funding.

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Despite a substantial advance in cancer treatment, pancreatic ductal adenocarcinoma (PDAC) have a limited response to current treatments, and a low 5-years survival rate of about 6% [1–3]. Thus, there is an urgent need to explore new mechanisms for treating this lethal malignancy.

Recent reports have discovered the capability of phenanthrenes to kill human cancer cells that are resistant to currently prescribed apoptosis-inducing agents [4, 5]. Furthermore, we identified phenanthrenes acting as PARP1 inhibitors that efficiently eradicate a variety of human cancer cells without impairing benign cells [6–9].

Notably, their exclusive cytotoxic activity in human cancer cells was independent of, and un-related to PARP1 inhibition [7–11]. The phenanthrenes PJ34, TiqA and phenanthridinon (Phen) act as PARP1 inhibitors due to their binding potency to the nicotine-amide binding site in the catalytic domain of PARP1 [12, 13]. However, their PARP1 inhibition per-se does not impair nor eradicate human malignant cells, including pancreas cancer cells, PANC1 [7–9].

In contrast, at higher concentrations than those causing PARP1 inhibition, PJ34, Tiq-A and Phen eradicate a variety of human cancer cells by ‘mitotic catastrophe cell death’. This cell-death follows mitosis arrest caused by preventing the post translational modification of NuMA (Nuclear mitotic apparatus protein-1) that enables its binding to proteins [8].

In the tested human cancer cells, NuMA binding to proteins enables its clustering in the spindle poles, which is crucial for stabilizing the spindle, a pre-requisite for chromosomes alignment in the spindle mid-zone and normal anaphase. Notably, NuMA silencing or down regulation of NuMA prevents mitosis in all cell types [14–17].

In human malignant cells, specific post translational modifications of NuMA enable and promote NuMA binding to proteins [18–21]. These post translational modifications are most efficiently prevented by PJ34 [8, 20, 21]. In accordance, in PJ34 treated cancer cells, NuMA is arbitrarily dispersed in the spindle, instead of being clustered in the spindle poles [8].

The consequences are un-stabilized spindle pole with dispersed chromosomes, instead of segregated chromosomes aligned in the spindle mid-zone [8, 17, 22, 23]. This abnormality jeopardizes normal ploidy of the ‘daughter’ cells and evokes mitosis arrest [17, 23]. Cells with these abnormal spindles are eradicated by a rapid death mechanism, ‘mitotic catastrophe’ cell-death [9, 23].

Here, the efficacy of PJ34 to eradicate human pancreas cancer cells is tested in cell cultures and in xenografts. PANC1 cells are most frequently identified in human pancreas tumors [1–3]. Pancreas xenografts were developed in nude mice.

These tumors also contained normal infiltrating cells (stroma) of mouse origin [24–27], i. e. fibroblasts, myofibroblasts, macrophages and lymphocytes infiltrated into the tumors [25, 26].

Mitosis arrest and cell death were measured in PANC1 cells incubated with PJ34. In xenografts, eradication of human PANC1 cells deduced from a massive reduction of human proteins in the tumors, was measured 30 days after the treatment with PJ34 has been terminated. An increased necrosis measured in the PANC1 tumors of mice treated with PJ34 supports cell-death caused by PJ34 in the xenografts. Normal cells infiltrated into the tumors were not impaired by PJ34. A similar cytotoxic activity of PJ34 was observed in patients-derived PDAC cells and xenografts. These results indicate the potency of PJ34 to eradicate human pancreas cancers.Go to:

RESULTS

Treatment with PJ34 causes mitosis arrest and cell death in human pancreas cancer cells PANC1

Measuring changes in the ploidy of PJ34 treated PANC1 cells with stained DNA by flow cytometry reveals piled up PANC1 cells with double DNA content, unable to proceed to mitosis (Figure 1). A similar cytotoxic effect of PJ34 is measured in other human malignant cell types [7]. Recently, the molecular mechanism causing mitosis failure and arrest in human cancer cells incubated with PJ34 has been disclosed [8].

The cell cycle profile of PANC1 cells incubated with PJ34 reflects their mitosis arrest and cell death (Figure 1). PJ34 (20 μM or 30 μM) was applied 24 hour after seeding, and PANC1 cell eradication and the kinetics of S-phase entry and G2/M transition were measured by flow cytometry after 48, 72 and 120 hours incubation (Methods).

After 48 hours incubation with PJ34, failure to proceed into mitosis preceded cell death measured after 72 hours incubation. These cells were eradicated after 120 hour incubation with PJ34 (Figure 1).

PJ34 efficacy tested in human PANC1 xenografts

PJ34 evoked cell death in PANC1 cells has been further examined in PANC1 xenografts. The course of the experiment was based on previous experiments with PANC1 xenografts [28]. A total of 24 nude mice were randomly divided into 3 groups of 8 mice each. PANC1 cells in cell cultures were subcutaneously injected (5 × 10⁶ cells per mouse; Methods).

Treatment with PJ34 started once PANC1 tumors reached a mean volume of approximately 100 mm³ (about 15 days after mice were injected with PANC1 cells). Nude mice of one group were injected intravenous (IV) with saline, daily 5 days a week for 3 weeks (control group). In the second group, nude mice were injected IV with PJ34 dissolved in saline, daily 5 days a week for 3 weeks. In the third group, nude mice were injected IV with PJ34 in saline, 3 times a week, every second day, for 3 weeks. Each injected dose contained 60 mg/Kg PJ34 in 100 μl saline (about 1 mg PJ34 per mouse). The volume of the developing tumors and the mice’ weight were monitored throughout the experiment (Figure 2A).

In previously described experiments with PANC1 xenografts, PANC1 tumors were developed during 60 days [28]. In our experiments, on day 63 of the study, and 30 days after terminating the treatment with PJ34, mice were euthanized. Their excised tumors were measured and prepared for immuno-histochemichemistry. During the 30 days after treatment with PJ34 has been terminated, mice were not treated with any additional treatment.

No abnormalities, toxic signs, or animal death were observed during the study. On the contrary, all mice gained weight during the study, with no difference between control and the two PJ34 treated groups, evidence for wellbeing of the treated mice (Figure 2A). In support, no toxicity has been found in an additional study with immunocompetent BALB/C mice, IV injected with even higher doses of PJ34. These mice were examined for 14 days after treatment. No signs of toxicity were detected, and PJ34 did not impair their weight gain (Supplementary Figure 1).

At the end of the study with the immunodeficient (nude) mice, only the group treated daily with PJ34 (5 times a week) showed a significant reduction of about 40% in tumor size (volume and weight, Figure 2B). In addition, in one treated mouse in this group, the tumor started to shrink on day 35 and disapeared on day 56 (Figure 2B, mouse #19). Furthermore, immunohistochemistry conducted in the excised tumors of all the tested mice revealed a massive reduction in human proteins and increasing necrosis in the tumors of mice treated with PJ34.

The excised tumors were sliced and prepared for histochemical analysis (Methods). Hematoxylin and Eosin labeling of the slices revealed higher necrosis in the tumors developed in PJ34 treated nude mice (Figure 3). In addition, the amounts of arbitrarily selected three human proteins were measured 30 days after the treatment with PJ34 has been terminated (Figure 4).

Immunolabeling of these human proteins in all the tumors revealed that both treatment regiments with PJ34 (daily and 3 times a week) caused 80–90% reduction in their amount in the tumors. The daily treatment provided better results with higher statistical significance (Figure 4E).

PANC1 cells were the only human cells in the PANC1 xenografts. Thus, the similar and substatial reduction in the amount of three arbitrarily selected human proteins, 30 days after the treatment with PJ34 has been terminated is attributed to the eradication of PANC1 human cells in the tumors of nude mice treated with PJ34 (Figure 4).

This is supported by the enhanced necrosis in tumors of mice treated with PJ34, and by the massive eradication of PANC1 cells incubated with PJ34 (Figures 1, ​,33 and [7, 8]). The relatively short turnover of proteins in the cell (hours), as well as the MRT (mean residence time, 41 min) of PJ34 in mice contradict a possible effect of PJ34 on the expression of the three arbitrarily selected human proteins, 30 days after the treatment with PJ34 has been terminated.

In addition, there was no reduction in the amount of an abundant protein in fibroblasts infiltrated into tumors of mice treated with PJ34, also contradicting an assumption of some general effect of PJ34 on protein expression 30 days after the treatment with PJ34 has been terminated.

The specific immunolabeling of each of the three proteins was measured in 3-4 different slices of each tumor, immunolabeled with specific antibody (Methods). Antibodies were directed against the human kinesin HSET/KifC1, or the c-terminal residue of the human nuclear protein Ku-80, or the Human Leucocytes Antigen (HLA), frequently found on the membrane of human cancer cells (Figure 4A–4D). Measurements of PJ34 induced changes in the immune-labeling of the three proteins provided results with a higher statistical significance. Normal cells infiltrated into the pancreas tumors (stroma) were immuno-labeled against smooth muscle actin αSMA that is mainly expressed in normal fibroblasts of both human and rodents [24, 25] (Figure 4C). The benign cells infiltrating into the human PANC1 xenografts were probably of mouse origin [26, 27]. In each slide, 6–8 different ‘fields’ were analysed (Methods; Figure 4E). Abnormal human tissues that may comprise metastasis were not found in the liver or guts, neither in treated nor in the untreated control mice. This fact is in accordance with other signs indicating the wellbeing of all the mice in this study (Figure 2A).

PJ34 efficacy tested in patients-derived pancreas cancer cells and xenografts

In view of evidence indicating eradication of PANC1 cancer cells by PJ34 (Figures 1 and ​and4),4), the effect of PJ34 was further tested in other types of patients-derived pancreas cancer cells. Ascites/pleural effusion derived cells were prepared from 4 different deceased pancreas cancer patients in the Sheba Medical Center [1] (Methods).

These cells did not include PANC1 cells. Samples of the effusion fluid were injected subcutaneously in nude mice, and cell cultures were prepared from the developed tumors. PJ34 (15 and 30 μM) added to these cell cultures, 24 hours after seeding has been tested for its effect on cell viability during 24, 48, 72 and 96 hours incubation.

The effect of PJ34 was measured by the Sulforhodamine B (SRB) cytotoxicity assay (Methods). PJ34 dose-dependently reduced the cell counts in the four different patients-derived cell cultures (Figure 5A). Next, PJ34 has been tested in xenografts developed after injecting nude mice (8) with Acite/pleural effusion of pancreas cancer patient #1.

After pancreas tumors reached a volume of about 100 mm³. treatment with PJ34 started. Mice were injected daily with PJ34 (I. P.) 5 days a week for 3 weeks (60 mg/Kg PJ34, about 1 mg PJ34 in 100 μl saline per mouse). Untreated control nude mice were similarly injected IP with saline. The study ended 5 days after terminating the treatment with PJ34. Both control and PJ34 treated mice were euthanized and their excised tumors were measured and prepared for immuno-histochemichemistry (Methods).

Human kinesin HSET/kifC1, HLA and human Ku-80 were specifically immunolabeled in all the tumors. About 90% reduction in the amount of HSET/kifC1 (p = 0.00067) and in Ku-80 (p = 0.00002) were measured in tumors developed in mice treated with PJ34 (Figure 5B). It was attributed to the eradication of the patients-derived pancreas cancer cells in the xenografts. Notably, treatment with PJ34 caused a similar reduction in HSET/KifC1 and Ku-80 labeling in PANC1 xenografts and in xenografts of pancreas patient#1 (Figures 4E and ​and5B5B).