Tumor Multiplicity In Mice Research Articles

Abstract 2596: Stage-specific inhibition of NNK-induced lung carcinogenesis by 1,4-phenylenebis(methylene)seleno-aspirin (p-XS-Asp)

Abstract Chemopreventive efficacy of p-XSC is well documented but it failed due to systemic toxicity issues. This toxicity could partially be due to the release of poisonous hydrogen cyanide generated after p-XSC metabolizes to form active bis-selenol (p-XSeH). To address the said concern, we recently designed and developed p-XS-Asp, with the rationale that it would cleave in vivo to release the active p-XSeH and aspirin instead of undesired HCN, thus making the compound less toxic and possibly more potent than p-XSC. Indeed, we have shown previously that p-XS-Asp inhibits NNK-induced lung tumorigenesis in A/J mice more effectively than p-XSC in a complete A/J mouse model, and is better tolerable. The complete model, however, does not reveal at what stage of carcinogenesis does p-XS-Asp acts. In the current study, we evaluated the stage-specificity of chemopreventive efficacy of p-XS-Asp in NNK-induced lung carcinogenesis in A/J mice models. A/J mice were divided into 7 distinct groups (n=30 per group, half male, half female; except group 1 and 2 where n=10). The mice were fed AIN-93M diet (control) or p-XS-Asp diet until they reached termination end point of 26 weeks (adenomas) and 40 weeks (adenocarcinomas). Two weeks after the experiment started, all the groups, except for group 1 and 2, were given a single IP injection of 10 µmol (100 mg/kg) of NNK. Group 3 and 4 were on complete control AIN93M and p-XS-Asp diets, respectively. Group 5 (peri-initiation) was on p-XS-Asp for the first 3 weeks and then on the control diet till the end of the experiment. On the other hand, group 6 (post-initiation) was on control diet for 3 weeks and then was changed to the p-XS-Asp diet. In the progression group 7, mice were on control diet for 14 weeks and subsequently changed to p-XS-Asp diet. The Groups 4 and 5 mice showed a remarkable decrease in the tumor multiplicity (TM) and incidence (TI) as compared to the mice on control diet at both 26 and 40 week time-points. The TM in male mice for groups 1, 2, 3, 4, 5, 6 and 7 at 26 weeks was 0.6, 0, 5.7, 1, 1.54, 4.27, and 6.27, respectively, while at 40 weeks, it was 0.2, 0, 7.7, 1, 1.8, 4.1, and 4.9, respectively. A similar trend was also observed in female mice. These data clearly demonstrated that robust inhibition at peri-initiation stage is mainly responsible for the activity observed in the complete model. The inhibition of both O6-methylguanine and pyridoxobutyl mutagenic DNA adducts by p-XS-Asp further compliments that the compound acts at initial stages of carcinogenesis. Body weights comparison and the blood and tissue analyses showed no systemic toxicity for the p-XS-Asp fed groups. RNA-seq data of the tumor tissues showed numerous signaling pathways to be affected in p-XS-Asp treated mice. The exact mechanism of action is still under investigation. In summary, our results show that p-XS-Asp may be promising candidate for future clinical evaluation as a lung cancer preventive agent. Citation Format: Asif Raza, Amandeep Singh, Cesar Aliaga, Daniel Plano, Shantu Amin, Arun K. Sharma. Stage-specific inhibition of NNK-induced lung carcinogenesis by 1,4-phenylenebis(methylene)seleno-aspirin (p-XS-Asp) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2596.

Abstract 14: TRAIL inducing small molecule ONC201 prevents intestinal tumors in FAP mouse model

Abstract Colorectal cancer (CRC) is a major health problem globally. Preventing CRC in the general population, and especially in high-risk cohorts with colonic polyps, or its recurrence, is a high priority. ONC201, an orally active small molecule with robust antitumor activity, is currently in clinical trials for advanced cancers. The aim of this study was to determine the CRC preventive potential of ONC201 using the Apcmin/+ mouse model for CRC in familial adenomatous polyposis. First, the optimal and non-toxic doses of ONC201 were determined. C57BL/6J mice (n=6/group) were randomized, and five different doses of ONC201 (0-100mg/kg BW) were administered orally twice a week by gavage. After six weeks of treatment, all mice were euthanized, and tissues were analyzed for optimal dose and toxicity. Based on the body weight gain, gross organ weights at termination, blood profiling, plasma liver enzyme profile, and histopathological analysis, it was concluded that there were no toxicities associated with ONC201 within the tested dose range. For efficacy evaluation, Apcmin/+ male and female mice (n≥20) were grouped and ONC201 (0, 25 and 50 mg/kg BW) was administered by gavage, twice weekly for 14 weeks. At termination, intestinal tumors were evaluated for incidence and multiplicity to determine the efficacy of ONC201. Results indicate a strong suppressive effect of ONC201 against colonic and small intestinal (SI) tumor multiplicity in mice of both genders. Colonic tumor incidence was significantly reduced by >50% in ONC201-treated (50 mg/kg BW) male (40% in treated vs. 92% in control; p<0.0002) and female mice (30% in treated vs. 81% in control; p<0.001) when compared with placebo. Colonic tumor multiplicity was reduced by 68% in ONC201-treated (50 mg/kg BW) male mice (0.44 ± 0.11 in treated vs. 1.4 ± 0.14 in control; p<0.0001) and by 75% in female mice (0.30 ± 0.10 in treated vs. 1.19 ± 0.19 in control; p<0.0003). SI polyps were suppressed by up to 68% in both male mice (11.40 ± 1.19 in treated vs. 36.08 ± 2.62 in control; p<0.0001) and female mice (9.65 ± 1.15 in treated vs. 29.24 ± 2.51 in control; p<0.0001). Snap-frozen colon tumor samples were analyzed for various biomarkers. Western blot protein analysis of the tumors suggested a dose-dependent increase in TRAIL expression upon treatment with ONC201. Similar increases were observed for DR5 and cleaved caspase 8 protein expression. Cleaved caspase 7, caspase 3, FADD and p21 were found to be increased in the treated tumor samples when compared with control. Immunohistochemical analysis indicated a significant decrease in PCNA index in the ONC201-treated tumors compared with controls. Serum analysis indicated a decrease in pro-inflammatory biomarkers, such as IL1β, IL6, TNFα, G-CSF, and GM-CSF, in the ONC201-treated samples compared to controls. Overall, chronic oral administration of ONC201 demonstrated strong chemopreventive efficacy against intestinal tumorigenesis in the Apcmin/+ mouse model. (Supported by NCI HHSN 261201500038I) Citation Format: Chinthalapally V. Rao, Venkateshwar Madka, Yuting Zhang, Gopal Pathuri, Janani Panneerselvam, Nicole Stratton, Niklas K. Finnberg, Howard P. Safran, Jennifer Fox, Shizuko Sei, Elizabeth R. Glaze, Robert Shoemaker, Wafik S. El-Deiry. TRAIL inducing small molecule ONC201 prevents intestinal tumors in FAP mouse model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 14.

Inhalation of iron-abundant gas metal arc welding-mild steel fume promotes lung tumors in mice

Welding fumes were reclassified as a Group 1 carcinogen by the International Agency for Research on Cancer in 2017. Gas metal arc welding (GMAW) is a process widely used in industry. Fume generated from GMAW-mild steel (MS) is abundant in iron with some manganese, while GMAW-stainless steel (SS) fume also contains significant amounts of chromium and nickel, known carcinogenic metals. It has been shown that exposure to GMAW-SS fume in A/J mice promotes lung tumors. The objective was to determine if GMAW-MS fume, which lacks known carcinogenic metals, also promotes lung tumors in mice. Male A/J mice received a single intraperitoneal injection of corn oil or the initiator 3-methylcholanthrene (MCA; 10 μg/g) and, one week later, were exposed by whole-body inhalation to GMAW-MS aerosols for 4 hours/day x 4 days/week x 8 weeks at a mean concentration of 34.5 mg/m3. Lung nodules were enumerated by gross examination at 30 weeks post-initiation. GMAW-MS fume significantly increased lung tumor multiplicity in mice initiated with MCA (21.86 ± 1.50) compared to MCA/air-exposed mice (8.34 ± 0.59). Histopathological analysis confirmed these findings and also revealed an absence of inflammation. Bronchoalveolar lavage analysis also indicated a lack of lung inflammation and toxicity after short-term inhalation exposure to GMAW-MS fume. In conclusion, this study demonstrates that inhalation of GMAW-MS fume promotes lung tumors in vivo and aligns with epidemiologic evidence that shows MS welders, despite less exposure to carcinogenic metals, are at an increased risk for lung cancer.

Ultraviolet radiation accelerates NRas-mutant melanomagenesis: A cooperative effect blocked by sunscreen.

To mitigate melanoma risk, sunscreen use is widely advocated; yet, the ability of sunscreens to prevent melanoma remains controversial. Here, we test the tenet that sunscreens limit melanoma risk by blocking ultraviolet radiation (UV)-induced DNA damage using murine models that recapitulate the genetics and spontaneous evolution of human melanoma. We find that a single, non-erythematous dose of UV dramatically accelerates melanoma onset and increases tumor multiplicity in mice carrying an endogenous, melanocyte-specific NRas61R allele. By contrast, transient UV exposure does not alter tumor onset in mice lacking p16INK4a or harboring an NRas12D allele. To block the rapid onset of melanoma cooperatively caused by UV and NRas61R , we employed a variety of aerosol sunscreens. While all sunscreens delayed melanoma formation and blocked UV-induced DNA damage, differences in aerosol output (i.e., amount applied/cm2 ) caused variability in the cancer preventative efficacy of products with identical sunburn protection factor (SPF) ratings.

Abstract 1605: α-cryptoxanthin supplementation inhibits the carcinogen-initiated and nicotine-promoted lung carcinogenesis in AJ mice by suppression of AKT activation

Abstract A pooled analysis from well-implemented cohort studies reported that increased dietary α-cryptoxanthin (BCX) intake, rather than α-carotene used in earlier human trials, is associated with reduced risk of lung cancer. Nonetheless, BCX efficacy on lung carcinogenesis has not been reported. In these studies, we examined the protective effects of BCX at different doses against lung carcinogenesis at the initiation and the promotion stages of the cancer development in male AJ mice. In the tobacco carcinogen 4-(methylnitrosamino)- 1-(3-pyridyl)-1-butanone [NNK]-initiated lung carcinogenesis model, we found a 52-63% reduction of lung tumor multiplicity in mice pre-treated with BCX at 0.2 and 2 mg kg BW-1 d-1, as compared to the NNK group (P<0.01). In contrast, BCX did not significantly alter the spontaneous lung tumor multiplcitiy in mice without the NNK treatment. Furthermore, in the NNK-initiated and nicotine-promoted lung cancer model, we found that nicotine treatment by intraperitoneal injections not only significantly induced emphysema, a risk for lung cancer, but also greatly increased lung tumor multiplicity of the NNK-treated mice. Remarkably, BCX at 2 and 4 mg kg BW-1 d-1 prevented the nicotine-induced pulmonary emphysema and decreased tumor multiplicity by 86-91% (P<0.01). BCX protective effect on carcinogenesis was associated with significant suppression of lung AKT activation measured by immunoblotting. BCX also reduced the emphysema-related IL-6 and early growth response-1 induction of mRNA levels measured by real-time PCR. Contrary to α-carotene reported in prior animal studies, BCX also induced the retinoic acid receptor-α mRNA. As measured by HPLC analysis, the concentrations of liver BCX were higher in the BCX supplemented mice, without alteration of the retinol concentrations. Our data suggest that BCX is a unique chemopreventive agent for lung cancer prevention, which is likely to be independent of its provitamin A activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1605. doi:1538-7445.AM2012-1605

β‐Cryptoxanthin supplementation inhibits carcinogen‐initiated and nicotine‐promoted lung tumor development in AJ mice

Recent epidemiologic studies show that increased dietary β‐cryptoxanthin (BCX) intake, rather than β‐carotene used in earlier human trials, is associated with reduced risk of lung cancer. However, BCX efficacy on lung carcinogenesis has not been reported. Here, we report the protective effects of different doses of BCX against lung carcinogenesis at the initiation and promotion stages in AJ mouse models. In the tobacco carcinogen NNK‐initiated lung carcinogenesis model, we found a 52–63% reduction of lung tumor multiplicity in mice pre‐treated with BCX at 0.2 and 2 mg kg BW‐1 d‐1, as compared to the NNK group, P<0.01. In the NNK‐initiated and nicotine‐promoted lung cancer model, we found that nicotine not only induced emphysema, a risk for lung cancer, but also increased greatly lung tumor multiplicity of the NNK‐treated mice. Notably, BCX at 2 and 4 mg kg BW‐1 d‐1 prevented nicotine‐induced emphysema and decreased tumor multiplicity by 86–91%, P<0.01. BCX protection was associated with suppressions of lung AKT activation, IL‐6 and early growth response‐1 mRNA. Unlike β‐carotene reported in prior animal studies, BCX induced retinoic acid receptor‐β mRNA. By HPLC analysis, liver BCX, not retinol, increased in the BCX supplemented mice. Our data suggest that BCX is a unique chemopreventive agent for lung cancer prevention, which is likely to be independent of its provitamin A activity.Grant Funding Source : NIH R01CA104932

Abstract 858: Transplacental cancer prevention by indole-3-carbinol via estrogen receptor beta

Abstract Indole-3-carbinol (I3C), a key active component of cruciferous vegetables, was recently shown to be an effective anticancer agent when administered transplacentally to mice via the maternal diet. The chemopreventive properties of I3C can involve multiple mechanisms of action, including interaction with the aryl hydrocarbon receptor (AhR) and the estrogen receptor (ER). In this prior transplacental cancer study, the chemopreventive property of I3C was not dependent on the AhR. In the present study, the therapeutic efficacy of I3C in prevention of cancer in offspring exposed in utero to the environmental carcinogen dibenzo[a,l]pyrene (DBP) was evaluated using an ER beta knockout mouse model. B6129SF1/J-Esr2+/- dams and sires were crossed to generate offspring that were either wild-type, heterozygous or null for ER beta. Dams were initiated with 15 mg/kg DBP or sham (corn oil) on gestation day 17. Three experimental diets were evaluated as follows: group 1, dams and offspring were fed control diet throughout the study; group 2, dams were fed 2000 ppm I3C from gestation day 9 through lactation (I3C-GL); and group 3, offspring were fed 2000 ppm I3C from weaning until 6 months of age (I3C-PW). As previously observed, offspring in the DBP-initiated control group developed T-cell lymphoblastic lymphoma by 20 weeks of age, whereas mice in the I3C-GL group were less likely to succumb to the disease (p<0.0001). Although I3C did not reduce incidence of lung cancer in the surviving offspring, lung tumor multiplicity in mice exposed to I3C during gestation and lactation (I3C-GL) was significantly reduced compared to control (p<0.05). I3C administered directly to the offspring (I3C-PW) did not provide protection against DBP-dependent lymphoma or reduce lung tumor multiplicity. This observation points to the critical gestation/lactation window of exposure for chemoprevention by this compound. A significant effect of ER beta genotype was observed in offspring exposed to I3C during gestation and lactation, only. Lymphoma-related mortality was very low in offspring expressing one or both copies of ER beta (+/-, 89% survival; +/+, 96% survival), whereas mortality in offspring lacking ER beta was significantly higher (−/-, 58% survival; p=0.0001.) Interestingly, in this diet group, lung tumor multiplicity in ER beta null offspring was significantly lower than wild-type or heterozygous mice. Collectively, these findings suggest that I3C acts via ER beta to exert its anticancer effects when administered transplacentally to offspring via the maternal diet. Supported by grants F32ES014777, P01CA090890 and the Linus Pauling Institute. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 858. doi:10.1158/1538-7445.AM2011-858

Identifying efficacious approaches to chemoprevention with chlorophyllin, purified chlorophylls and freeze-dried spinach in a mouse model of transplacental carcinogenesis

The carcinogenic potential of dibenzo[a,l]pyrene (DBP) has been well characterized in numerous animal models. We have previously documented that a single dose of 15 mg/Kg DBP to pregnant mice late in gestation (GD 17) produces an aggressive T-cell lymphoma as well as lung and liver cancer in offspring. The current study examines the chemopreventative properties of chlorophyllin (CHL) and chlorophyll (Chl) in this transplacental carcinogenesis model. Pregnant B6129SF1 females, bred to 129S1/SvIm males, received purified diets incorporated with either 2000 p.p.m. CHL, 2000 p.p.m. Chl or 10% freeze-dried spinach beginning at gestation day 9. Lymphoma-dependent mortality was not significantly altered by maternal consumption of any of the diet and little effect on lung tumor burden in mice surviving to 10 months of age was observed. However, coadministration of CHL at 380 mg/Kg with DBP by gavage (molar ratio of 10:1, CHL:DBP) provided significant protection against DBP-initiated carcinogenesis. Offspring born to dams receiving CHL co-gavaged with DBP exhibited markedly less lymphoma-dependent mortality (P < 0.001). The degree of protection by CHL, compared with controls dosed with DBP in tricaprylin (TCP) as the vehicle, was less marked, but still significant. Coadministration of CHL (TCP as vehicle) also reduced lung tumor multiplicity in mice by approximately 50% and this was observed throughout the study (P < 0.005). This is the first demonstration that CHL can provide potent chemoprotection in a transplacental carcinogenesis model and support a mechanism involving complex-mediated reduction of carcinogen uptake.

Strain-dependent lung tumor formation in mice transplacentally exposed to 3-methylcholanthrene and post-natally exposed to butylated hydroxytoluene.

The carcinogenic effects of in utero exposure to 3-methylcholanthrene (MC) have been demonstrated in the tumor-resistant C57BL/6 (B6) and DBA (D2) strains of mice. In this study, we determined the effects of in utero exposure to MC in BALB/c mice, a strain which demonstrates greater susceptibility to lung tumor induction, and compared our findings with those previously found in [D2xB6D2F(1)]F(2) mice. In addition, we assessed the molecular pathogenesis of the chemically induced tumors and examined the effects of the putative lung tumor promoter butylated hydroxytoluene (BHT) in BALB/c mice. BALB/c mice were treated on day 17 of gestation with 5, 15 or 45 mg/kg MC and 6 weeks after birth with BHT for 6 consecutive weeks. Mice were killed at 6 months of age. Ki-ras, p16Ink4a and p19ARF gene loci were amplified from paraffin-embedded lung tumor tissue and screened for the presence of point mutations via allele-specific oligonucleotide hybridization and single strand conformation polymorphism (SSCP) analyses. Ki-ras point mutations were found in 56% (20/36) of BALB/c lung tumors, with 33% (2/6) of the hyperplasias, 58% (10/19) of the adenomas and 73% (8/11) of the carcinomas exhibiting point mutations at this gene locus. Similar incidences of Ki-ras mutations were previously found following transplacental exposure of [D2xB6D2F(1)]F(2) mice to MC and treatment of adult A/J mice with urethane. Interestingly, a strain-dependent difference was observed in the mutational spectrum. Sixty-two and 38% of the lung lesions in BALB/c mice exhibited G-->C and G-->T transversions, respectively, in contrast to the 13 and 84% incidences previously observed in [D2xB6D2F(1)]F(2) mice. SSCP analysis of the tumor suppressor gene p16Ink4a showed a 6% incidence of point mutations, consistent with that found in [D2xB6D2F(1)]F(2) mice. No mutations were found in exon 1beta of the p19ARF gene of either strain. BHT, a lung tumor promoter in adult mice, had no statistically significant effects on either tumor incidence, tumor multiplicity or the mutational spectrum produced in the Ki-ras gene by in utero MC treatment. However, though not significant, there was an observable trend in increased tumor multiplicity in mice co-treated with BHT. These data demonstrate the transplacental carcinogenic effect of MC in BALB/c mice and show that mutagenic damage to Ki-ras is a critical early event mediating murine lung tumorigenesis in both the tumor-sensitive and tumor-resistant strains. Unlike what occurs when adult BALB/c mice are treated with MC, BHT does not appear to significantly promote the formation of lung tumors following transplacental exposure to MC, possibly due to the rapid growth and cell proliferation in the developing organism. Strain-dependent differences in the Ki-ras mutational spectrum may be associated with their differential susceptibility to lung tumor initiation.

Inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone pulmonary metabolism and tumorigenicity in mice by analogues of the investigational chemotherapeutic drug 4-ipomeanol.

4-Ipomeanol (IPO) is an investigational chemotherapeutic drug with specific toxicity toward the lung. It is metabolically activated to reactive intermediates by cytochrome P450 enzymes present in Clara cells. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a highly carcinogenic tobacco-specific nitrosamine with organo-specificity for the lung. Like IPO, which it resembles structurally, it is metabolically activated by cytochrome P450 enzymes of rat Clara cells. We synthesized nontoxic analogues of IPO and tested their activities as inhibitors of the metabolism and tumorigenicity of NNK. The IPO analogues synthesized were 4-hydroxy-1-phenyl-1-pentanone (HPP), 7-hydroxy-1-phenyl-1-octanone (HPO), 4-hydroxy-1-(2-thienyl)-1-pentanone (HTP), and 4-hydroxy-1-(3-pyridyl)-1-pentanone (HPYP). When added to A/J mouse lung microsomal incubations, all compounds significantly inhibited the oxidative pathways of NNK metabolism--alpha-hydroxylation and pyridine N-oxidation--to varying extents. Inhibition of carbonyl reduction of NNK was generally less effective. Inhibition of alpha-hydroxylation by IPO, HPP, and HTP was more pronounced in incubations with lung microsomes than with liver microsomes. None of the IPO analogues showed significant toxicity when given to A/J mice at a dose of 25 mumol; IPO itself was lethal at this dose. HPP and HPO, at doses of 25 mumol, significantly inhibited lung tumor multiplicity in mice treated with NNK; the other analogues and IPO itself were ineffective. The results of this study provide new leads for development of inhibitors of NNK metabolism and chemical probes for the active site of P450 enzymes in Clara cells.

Rapid single-dose model for lung tumor induction in A/J mice by 4-(methylnitrosainino)-1-(3-pyridyl)-1-butanone and the effect of diet

This paper describes the development of a relatively rapid single-dose model for induction of lung adenomas in female A/J mice by the tobacco-specific nitrosamine 4-(methylnitros-amino)-1-(3-pyridyl)-1-butanone (NNK). Mice maintained on AIN-76A semi-synthetic diet were given a single i.p. dose of 2.5, 5 or 10 mumol NNK in saline and killed 3-7 months later. Maximum lung tumor induction, measured by lung tumors per mouse (tumor multiplicity), occurred in 3.5 months. There was no significant increase in tumor multiplicity between 3.5 and 7 months. Four months after treatment, numbers of lung tumors per mouse were 11.9 +/- 1.0 (10 mumol NNK), 3.6 +/- 0.4 (5 mumol), 0.9 +/- 0.4 (2.5 mumol) and 0.07 +/- 0.1 (saline). Lung tumor multiplicity in mice treated with a single dose of 10 mumol NNK and maintained on AIN-76A diet was significantly higher (8.3 +/- 0.5) than in mice treated with NNK and maintained on NIH-07 diet (2.5 +/- 0.3). The results of this study establish a useful bioassay for identification of compounds that can modify NNK-induced lung tumorigenesis.