Inhibitory Effect of (2R)-1-(1-Benzofuran-2-yl)-N-propylpentan-2-amine on Lung Adenocarcinoma
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BPAP is a potent enhancer substance with catecholaminergic and serotoninergic activity in the brain. It was discovered that it is also effective against certain types of experimental cancers, showing the most promising results in case of lung cancer. That is why we tested its efficacy in two different doses in a newly developed EGFR wild type mouse lung adenocarcinoma xenograft model. Experiments were conducted on FVB/N and SCID mouse strains treated with low and high dose of BPAP. Body weight, survival, and tumor volumes were recorded. Furthermore, the activity of major signaling pathways of NSCLC such as MAPK and Akt/mTOR as well as cell cycle regulation were determined. Significant inhibition of tumor growth was exerted by both doses, but the mechanism of action was different. High dose directly inhibited, whereas low dose activated the main signaling pathways. Exposure to low dose BPAP resulted in elevated activity of the mTOR pathway together with p16INK-induced cell cycle arrest, a typical feature of geroconversion, a senescent state characterized by loss of cell proliferation. Finally the events culminated in cell cycle inhibition point in case of both doses mirrored by the decrease of cyclin D1, CDK4 and PCNA. In addition, BPAP treatment had a beneficial effect on bodyweight suggesting that the compound at least in part is able to compensate the cancer-related wasting. In view of the low toxicity and confirmed antitumor effect of BPAP against experimental lung adenocarcinoma, this novel compound deserves further attention.
KeywordsBPAP Lung adenocarcinoma Cancer Tumor inhibition FVB/N Geroconversion
protein kinase B
anaplastic lymphoma kinase
cyclin-dependent kinase 4
ethylene diamine tetraacetic acid
epidermal growth factor receptor
Kirsten Rat Sarcoma virus oncogene
mouse double minute 2 homolog
mechanistic target of rapamycin
non-small cell lung cancer
poly ADP ribose polymerase
ploriferating cell nuclear antigen
severe combined immunodeficiency
The phenethylamine derivative (−)-deprenyl (also known as selegiline) gained attention in the 1970s when its ability to block MAO-B enzyme  was confirmed and was hence the first to be used in the treatment of Parkinson’s disease . Longevity studies showed that deprenyl can prolong the lifespan of rats, mice and beagle dogs when given in a daily dose of 0.25 mg/kg [3, 4, 5]. Later on 1-phenyl-2-propylaminopentane (PPAP) was synthetized, lacking the MAO inhibitory effect but still being a potent enhancer of catecholaminergic signaling in the central nervous system . Based on above results, a tryptamine derived new compound, (2R)-1-(1-Benzofuran-2-yl)-N-propylpentan-2-amine (BPAP) was created, capable of potentiating catecholaminergic and serotonergic activity in the brain . BPAP has been shown to have neuroprotective and memory stimulatory effects similar to those of deprenyl but without the ability to inhibit the MAO-B enzyme .
Both BPAP and deprenyl are capable of exerting their effects in a very low concentration range . The impact on learning skills and lifespan of long-term deprenyl and BPAP treatment at low concentration ranges was monitored in a longevity study carried out on Wistar rats. Both molecules exhibited a lifespan-extending effect, but BPAP showed more promising results . It was noticed that significantly less spontaneous fibrosarcomas developed in the treated groups than in the control group . Based on these observations, we tested the effects of low-dose BPAP treatment in different tumor models.
In an in vivo model of colon cancer with liver metastasis, BPAP significantly reduced the number of metastasis . In addition, BPAP was tested in other tumor models as well: xenograft models of mouse colon carcinoma and lung adenocarcinoma and in a primary hepatocarcinogenesis experiment induced by diethylnitrosamine in mice. The most promising results were obtained in the lung tumor model established from a FVB/N strain, in which lung adenocarcinomas developed after exposure to single doses of diethylnitrosamine .
Lung cancer is the most common cancer in Europe and the United States claiming the largest number of lives [13, 14]. Histologically, the disease is divided into two major classes: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) . The latter is the more common and in the United States 85% of lung cancers are NSCLC . In the last decade, adenocarcinomas became the dominant representative within NSCLC .
It is not unprecedented that a molecule effective in a non-malignant disorder is found to inhibit certain types of cancers . Several nonsteroidal anti-inflammatory drugs, statins and other types of anti-psychotic drugs were shown to have anti-tumor effect .
In the present work, we aimed to examine the effects and mechanism of action of BPAP in lung adenocarcinomas.
Materials and Methods
Animals and Treatments
All animal experiments were conducted according to the ethical standards of the Animal Health Care and Control Institute of Csongrád County, Hungary and the protocol was approved by their Committee (permit No. XVI/03047–2/2008). Animals were obtained from the animal facility of the 1st Department of Pathology and Experimental Cancer Research of Semmelweis University (Budapest, Hungary).
To examine the effects of BPAP on lung adenocarcinoma, a subcutaneous mouse tumor model was applied . To this end, a total of 45 FVB/N male mice at the age of 8 weeks were utilized, 27 in Experiment 1 and 18 in Experiment 2. Animals were treated on a daily basis with 0.0001 mg/kg (low dose) or 0.05 mg/kg (high dose) BPAP starting from the day after subcutaneous tumor inoculation. The control group received saline once every day. Tumors were measured 3 times a week by the same investigator using a digital caliper. Tumor volume was calculated using the following formula: V (mm3) = (width2 (mm) × length (mm) × π)/6. Mice were terminated by cervical dislocation in ether anesthesia. Tumors were removed, their weight measured, were stored at −80 °C. The experiment was repeated on SCID mice to determine whether the adaptive immune system has a role in the tumor suppressor effect of BPAP (Experiment 3). Eighteen animals were utilized in this 3rd study, and were terminated after 28 days.
Deprenyl was supplied by Sanofi-Chinoin (Budapest, Hungary) and BPAP by Fujimoto Pharmaceutical Corporation (Osaka, Japan).
Frozen tumor tissues were homogenized and suspended with lysis buffer (containing: 20 mM Tris pH = 7.5, 150 mM NaCl, 2 mM EDTA, 0.05% Triton X-100, 0.5% Protease Inhibitor Cocktail (P8340, Sigma-Aldrich, St. Louis, MO), 2 mM Na3VO4 and 10 mM NaF). Samples were sonicated and kept on ice before centrifugation at 13000 rpm at 4 °C for 10 min. Protein concentrations were measured by Bradford method . Thirty μg total proteins were mixed with loading buffer containing β-mercaptoethanol and denatured at 99 °C for 5 min.
Denatured samples were loaded onto a 10% SDS-polyacrylamide gel and separated for 40 min at 200 V. Proteins were transferred to a PVDF membrane with overnight blotting at 4 °C at a constant 75 mA. Successful blotting was confirmed by Ponceau staining. Blocking procedure was carried out with 5% non-fat dry milk dissolved in TBS. The primary antibodies applied with their appropriate dilutions are listed in Supplementary Table 1 . For negative control, 1% non-fat dry milk (in TBS) without primary antibody was applied. Incubation was overnight at 4 °C with gentle shaking. Secondary antibody was anti-rabbit or anti-mouse polyclonal HRP-conjugated antibody (Dako, Glostrup, Denmark) dissolved in 1% non-fat dry milk (in TBS) applied for 1 h at room temperature with gentle shaking. For washing 0.05% Tween20 in TBS was used for 5 × 5 minutes between each step. For detection, SuperSignal West Pico Chemiluminescent Substrate (34,078, Thermo Fischer Scientific, Waltham, MA) was applied. Bands were detected with Kodak Image Station 4000 mm (Kodak, Rochester, NY). The bands were evaluated with GelAnalyzer program.
All statistical analyses were performed using GraphPad Prism 7.00 software (Graphpad Software Inc., La Jolla, CA, USA). Data were tested for normal distribution using the omnibus normality test of D’Agostino & Pearson. Significance of changes were tested using a nonparametric test (the Mann–Whitney U-test) or the Student’s t test, depending on the distribution of the data. The independent experimental sets were compared for reproducibility. Only reproducible significant changes were considered as significant. Significance was declared at the standard p < 0.05 level.
BPAP Inhibits Tumor Growth
Changes in Signaling Pathways Provoked by BPAP
BPAP against Cancer-Related Wasting
In the past decade, the detection of targetable somatic mutations has become essential in the treatment protocol of patients with non-small cell lung cancer . The mutations discovered in the tyrosine kinase domain of EGFR and ALK gene rearrangements, as therapeutic targets have fundamentally changed the treatment of NSCLC [19, 20]. However, these alterations are responsible for only a proportion of NSCLC cases, so only conventional chemotherapies remain for patients without targetable mutations .
Earlier, our research group established and characterized an in vivo mouse NSCLC tumor model system, with wild type KRAS and EGFR genes, mimicking the majority of human lung adenocarcinomas . It came as a surprise that when these tumors were exposed to BPAP the drug exerted a potent inhibitory effect on tumor growth. The substance was applied in a very low concentration range, with the low dose being 0.0001 mg/kg and the high dose 0.05 mg/kg bodyweight. Both doses were found to effectively hinder the proliferation of subcutaneously growing tumors. Further, the action mechanism proved to be different in case of both the signaling pathways and the cell cycle arrest at G1/S restriction point. In addition, since the effectiveness was detectable in FVB/N and SCID strains as well, the adaptive immune system is likely not involved in the mechanism .
In spite of the different pathways provoked by low- and high-dose BPAP, the signaling events were finally culminated in cell cycle arrest. Both BPAP doses inhibited the cell cycle at G1/S restriction point since the levels of cyclin D1 and CDK4 became decreased, thus preventing the retinoblastoma from inactivation. The lower PCNA levels observable upon BPAP treatment also point to inhibition of cell proliferation.
In case of low-dose BPAP, the highly stimulated signaling pathways in contrast to the ineffective G1 phase and hindered tumor growth called attention to the mechanism of geroconversion. In this hyper-mitogenic type of arrest, the cell cycle is suppressed by CDK inhibitors (such as p21WAF1/CIP1 or p16INK4), but mTOR and MAPK pathways are still active pushing the cell to become hyperthophic, hyper-active and hyper-functional. Activation of mTOR in the presence of cell cycle arrest induces senescent morphology and the loss of ability to proliferate [27, 28, 29]. The fact that we observed an increased amount of p16 accompanied by a high level of mTOR activity supports our presumption that low-dose BPAP provoked geroconversion upon treatment (Fig. 6). Interestingly, similar effect was observed upon exposure to PARP inhibitor in case of liposarcoma and other tumors, however, upregulation of p21WAF1/CIP1 and proteosomal degradation of MDM2 were accompanied by downregulation of CDK4 [30, 31].
The question is, what is the mechanism of p16 upregulation? A recent publication provides evidence that the AP-1 factor c-jun is able to interfere with tumorigenesis by protecting the promoter region of p16INK4a from methylation, acting as a “bodyguard” after oncogenic transformation . As we detected strong Erk1/2 activation, with subsequent upregulation of c-jun upon low dose BPAP exposure, it is highly conceivable that c-jun is implicated in the upregulation of p16INK.
In contrast with the results observed after low-dose BPAP, the high dose decreased the level of c-jun. C-jun is found to be elevated in 30% of NSCLC cases and cannot be found in the normal cells of airways [33, 34].
Despite the similar outcome, our data show that BPAP in low and high doses utilizes different mechanisms of action, since the latter exerts direct inhibition of the main signaling pathways (Fig. 6). A further question therefore is, what resulted the activation of p-S6 after BPAP high-dose administration? [35, 36].
Cancer cachexia is a terminal state in which patients lose their body fat and muscle weight rapidly as compared with immune system failures and metabolic dysfunctions . Both low- and high-dose BPAP exposure had a beneficial effect on cachexia. High-dose BPAP showed better protection, saving 22% more of the animal’s body mass. NF-κB is a key regulator of the events implicated in the development of cachexia . NF-κB has a role also in chemotherapy resistance, thus its inhibition may potentiate the efficacy of the therapy .
BPAP is a potent enhancer substance related to certain signaling events in the central nervous system. In a longevity study, it was discovered that the substance is also effective against certain types of cancers, showing the most promising results in case of lung cancer. In our study, the effects of two different doses of BPAP were studied in a newly developed EGFR wild type mouse lung adenocarcinoma xenograft. Both doses showed good results in tumor and cachexia inhibition, although seemingly having different mechanisms of action. High dose of BPAP showed direct inhibition on the main signaling pathways, whereas the low dose seemed to initiate geroconversion. Elevated activity of the mTOR pathway together with p16INK-induced cell cycle arrest resulted in geroconversion, which is a senescent state accompanied by loss of cell proliferation.
In view of the low toxicity and confirmed antitumor effect of BPAP in case of experimental lung adenocarcinoma, this novel compound deserves further attention.
The authors would like to thank Fujimoto Pharmaceutical Corporation (Osaka, Japan) for the generous gift of BPAP substance, András Sztodola for his valuable help in the animal nursing and Elvira Kálé Rigóné for language correction. This work was supported by National Scientific Research Fund (grant no: 105763).
Data Availability Statement
The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.
This work was supported by National Scientific Research Fund (grant no: 105763).
Compliance with Ethical Standards
Conflicts of Interest
The authors declare no conflict of interest.
All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.
- 1.Knoll J, Magyar K (1972) Some puzzling effects of monoamine oxidase inhibitors. Adv Biochem Psychopharmacol 5:393–408Google Scholar
- 4.Freisleben HJ, Lehr F, Fuchs J (1994) Lifespan of immunosuppressed NMRI-mice is increased by deprenyl. J Neural Transm Suppl 41:231–236Google Scholar
- 6.Knoll J, Knoll B, Torok Z, Timar J, Yasar S (1992) The pharmacology of 1-phenyl-2-propylamino-pentane (PPAP), a deprenyl-derived new spectrum psychostimulant. Arch Int Pharmacodyn Ther 316:5–29Google Scholar
- 8.Maruyama W, Yi H, Takahashi T, Shimazu S, Ohde H, Yoneda F, Iwasa K, Naoi M (2004) Neuroprotective function of R-(−)-1-(benzofuran-2-yl)-2-propylaminopentane, [R-(−)-BPAP], against apoptosis induced by N-methyl(R)salsolinol, an endogenous dopaminergic neurotoxin, in human dopaminergic neuroblastoma SH-SY5Y cells. Life Sci 75:107–117CrossRefGoogle Scholar
- 11.Knoll J, Baghy K, Eckhardt S, Ferdinandy P, Garami M, Harsing LG Jr, Hauser P, Mervai Z, Pocza T, Schaff Z, Schuler D, Miklya I (2017) A longevity study with enhancer substances (selegiline, BPAP) detected an unknown tumor-manifestation-suppressing regulation in rat brain. Life Sci 182:57–64CrossRefGoogle Scholar
- 19.Chan BA, Hughes BG (2015) Targeted therapy for non-small cell lung cancer: current standards and the promise of the future. Transl Lung Cancer Res 4:36–54Google Scholar
- 20.Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW, Harris PL, Haserlat SM, Supko JG, Haluska FG, Louis DN, Christiani DC et al (2004) Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 350:2129–2139CrossRefGoogle Scholar
- 24.Yip PY (2015) Phosphatidylinositol 3-kinase-AKT-mammalian target of rapamycin (PI3K-Akt-mTOR) signaling pathway in non-small cell lung cancer. Transl Lung Cancer Res 4:165–176Google Scholar
- 30.Yoshida A, Diehl JA (2015) CDK4/6 inhibitor: from quiescence to senescence. Oncoscience 2:896–897Google Scholar
- 31.Kovatcheva M, Liu DD, Dickson MA, Klein ME, O'Connor R, Wilder FO, Socci ND, Tap WD, Schwartz GK, Singer S, Crago AM, Koff A (2015) MDM2 turnover and expression of ATRX determine the choice between quiescence and senescence in response to CDK4 inhibition. Oncotarget 6:8226–8243CrossRefGoogle Scholar
- 34.Szabo E, Riffe ME, Steinberg SM, Birrer MJ, Linnoila RI (1996) Altered cJUN expression: an early event in human lung carcinogenesis. Cancer Res 56:305–315Google Scholar
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